PROTOCOL

SOLiD® Total RNA-Seq Kit

Publication Part Number 4452437 Rev. B
Revision Date July 2011
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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PATENTS AND LICENSING NOTIFICATIONS
The SOLiD® Total RNA-Seq Kit is covered by US and foreign patents pending.
TRADEMARKS
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© 2011 Life Technologies Corporation. All rights reserved.

Part Number 4452437 Rev. B

July 2011
 Contents
About This Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Purpose of this protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety alert words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ CHAPTER 1 SOLiD™ Total RNA-Seq Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Purpose of the product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Materials and equipment required but not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ CHAPTER 2 Whole Transcriptome Library Preparation:

Standard Input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Guidelines for RNA sample type and amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Fragment the whole transcriptome RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Fragment the RNA using RNase III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Fragment the RNA by chemical hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Clean up the fragmented RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Assess the yield and size distribution of the fragmented RNA . . . . . . . . . . . . . . . . . . . . . . . . . 18
Typical results of fragmentation of whole transcriptome RNA . . . . . . . . . . . . . . . . . . . . . . . . . 19
Construct the amplified whole transcriptome library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Hybridize and ligate the RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Purify and size-select the cDNA: round 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Purify and size-select the cDNA: round 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Amplify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Purify the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Assess the yield and size distribution of the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Proceed with SOLiD™ System templated bead preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Typical size profiles of amplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Expected yields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Using a positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ CHAPTER 3 Whole Transcriptome Library Preparation: Low Input . 33

About the low input procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Fragment the low input whole transcriptome RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Guidelines for RNA sample type and amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Fragment the RNA using RNase III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Clean up the fragmented RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Assess the yield and size distribution of the fragmented RNA . . . . . . . . . . . . . . . . . . . . . . . . . 38
Construct the amplified low-input whole transcriptome library . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

SOLiD® Total RNA-Seq Kit 3

Contents

Hybridize and ligate the low input poly(A) RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Purify and size-select the cDNA: round 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Purify and size-select the cDNA: round 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Amplify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Purify the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Assess the yield and size distribution of the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Proceed with SOLiD™ System templated bead preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Typical size profiles of amplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Using a positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Typical results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ CHAPTER 4 Small RNA Library Preparation . . . . . . . . . . . . . . . . . . . . . 51

Prepare the starting material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Guidelines for obtaining small RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Assess the amount and quality of small RNA in your total RNA samples . . . . . . . . . . . . . . . . 52
Guidelines for enriching for small RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Enrich the sample for small RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Assess the quality and quantity of the small RNA-enriched sample . . . . . . . . . . . . . . . . . . . . . 54
Determine the input amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Construct the amplified small RNA library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Hybridize and ligate the RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Purify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Size select the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Expected lengths of the insert and PCR product according to excised cDNA length . . . . . . . 60
Amplify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Purify the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Assess the yield and size distribution of the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Proceed with SOLiD™ System templated bead preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Typical size profiles of amplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Using a positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■ APPENDIX A Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

How to order . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Optional materials and equipment not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
For whole transcriptome libraries (standard input) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
For whole transcriptome libraries (low input) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
For small RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
For whole transcriptome libraries (standard input): Gel-based size selection . . . . . . . . . . . . 69

4 SOLiD® Total RNA-Seq Kit

 Contents
■ APPENDIX B Supplemental Information . . . . . . . . . . . . . . . . . . . . . . . . . 71
Amplified library construction concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Hybridization and ligation to the Adaptor Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Reverse transcription and size selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
cDNA library amplification (single- or multiplex) and final cleanup . . . . . . . . . . . . . . . . . . . . . 72
Sequences of the SOLiD™ primers included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
SOLiD™ 5′ PCR primer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
SOLiD™ 3′ PCR primer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
About the RNA fragmentation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Gel-based whole transcriptome library preparation: Standard Input . . . . . . . . . . . . . . . . . . . . . . . . 74
Gel size selection for Whole Transcriptome workflow standard input . . . . . . . . . . . . . . . . . . . . . . . . 75
Purify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Size select the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Example of 1st round size selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Expected lengths of the insert and PCR product according to excised cDNA length . . . . . . . 79
Amplify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Purify the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Assess the yield and size distribution of the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Typical size profiles of amplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Second-round size selection of amplified cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Size-select the amplified cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Purify the amplified cDNA from the gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Using 2100 expert software to assess whole transcriptome libraries . . . . . . . . . . . . . . . . . . . . . . . . 88
Perform a smear analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Determine the median size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Analyze multiple peaks as one peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Small RNA enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Using 2100 expert software to assess small RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Review the median size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Perform a smear analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Determine the % miRNA library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

■ APPENDIX C PCR Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . 97

■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

SOLiD® Total RNA-Seq Kit 5

Contents

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Kit documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

6 SOLiD® Total RNA-Seq Kit

 About This Protocol

Purpose of this protocol

This protocol provides procedures for preparing whole transcriptome libraries or
small RNA libraries using the SOLiD™ Total RNA-Seq Kit.

Revision history

Revision Date Description

B 04/2011 • Added procedures for preparing whole transcriptome libraries

starting from low input amounts of poly(A) RNA.
• Added chemical fragmentation procedures for the preparation of
whole transcriptome libraries.
• Added bead-based cDNA size selection for whole transcriptome
libraries.

Safety information

IMPORTANT! Before using this product, read and understand the “Safety Information”
appendix in this document.

Safety alert words Four safety alert words appear in Life Technologies user documentation at points in
the document where you need to be aware of relevant hazards. Each alert word—
IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of
observation or action, as defined below:

IMPORTANT! – Indicates information that is necessary for proper instrument

operation, or accurate chemistry kit use.

CAUTION! – Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against
unsafe practices.

WARNING! – Indicates a potentially hazardous situation that, if not avoided,

could result in death or serious injury.

SOLiD® Total RNA-Seq Kit 7

About This Protocol
Safety information

DANGER! – Indicates an imminently hazardous situation that, if not avoided,

will result in death or serious injury. This signal word is to be limited to the most
extreme situations.

8 SOLiD® Total RNA-Seq Kit

 Chapter 1 SOLiD™ Total RNA-Seq Kit
Product information 1

SOLiD™ Total RNA-Seq Kit

Product information
Purpose of the Use the SOLiD™ Total RNA-Seq Kit (PN 4445374) to convert RNA transcripts
product expressed in a cell or tissue into a cDNA library for analysis on the Applied Biosystems
SOLiD™ Sequencing System:
• Whole transcriptome libraries (standard input): To prepare whole transcriptome
libraries starting from standard input amounts of poly(A) RNA (100–500 ng) or
rRNA-depleted total RNA (200–500 ng), see page 13.
• Whole transcriptome libraries (low input): To prepare whole transcriptome
libraries starting from low input amounts of poly(A) RNA (5–25 ng), see page 33.
• Small RNA libraries: To prepare small RNA libraries, see page 51.
When you use the SOLiD Total RNA-Seq Kit with one of the SOLiD™ RNA Barcoding
Kits, you can prepare barcoded libraries to enable sequencing of multiple samples in a
single, multiplexed, SOLiD System sequencing run. Sequencing of multiplexed
libraries is fully supported by the SOLiD 4 and 5500 Systems. Instructions for using the
SOLiD™ RNA Barcoding Kits are included in product inserts that come with the kits
and in this protocol.

Kit contents and storage

Sufficient reagents are supplied in the SOLiD Total RNA-Seq Kit to prepare cDNA
libraries from 12 samples for high-throughput sequencing with the SOLiD System.
Upon receipt of the SOLiD Total RNA-Seq Kit, immediately store the components at
–20°C. You may store the Nuclease-free Water at room temperature, 4°C, or –20°C.

Component Amount Cap

Nuclease-free Water 1.75 mL clear

10✕ RNase III Reaction Buffer 20 µL red
RNase III 20 µL red
SOLiD™ Adaptor Mix 30 µL green
Note: The SOLiD™ Adaptor Mix is not equivalent to Adaptor
Mix A or Adaptor Mix B in other kits.
Hybridization Solution 40 µL green
2✕ Ligation Buffer 150 µL green
Ligation Enzyme Mix 30 µL green
10✕ RT Buffer 50 µL yellow
dNTP Mix 500 µL white
SOLiD™ RT Primer 30 µL yellow

SOLiD® Total RNA-Seq Kit 9

 Chapter 1 SOLiD™ Total RNA-Seq Kit
1 Product information

Component Amount Cap

ArrayScript™ Reverse Transcriptase 20 µL yellow

10✕ PCR Buffer 660 µL white
AmpliTaq® DNA Polymerase 110 µL white
SOLiD™ 5′ PCR Primer 100 µL white
SOLiD™ 3′ PCR Primer 100 µL blue
WT Control RNA (1 µg/µL HeLa total RNA) 50 µL clear
Small RNA Control (1 µg/µL human placenta total RNA) 10 µL purple

Materials and To order Applied Biosystems products, go to [Link]. For

equipment Ambion or Invitrogen products, go to [Link]. For widely available
products, go to major laboratory suppliers (MLS). For optional materials and
required but not
equipment and more ordering information, see page 67.
included
Item Catalog no.
Applied Biosystems thermal cycler with heated lid, capable of holding See the Applied
0.2-mL tubes: Biosystems website
• Veriti® 96-Well Thermal Cycler
• GeneAmp® PCR System 9700
Agilent® 2100 Bioanalyzer™ Instrument Agilent G2938A
Microcentrifuge MLS
NanoDrop® Spectrophotometer Thermo Scientific
Pipettors, positive displacement or air-displacement MLS
Ambion® 8-strip PCR Tubes & Caps, RNase-free, 0.2-mL AM12230
Ambion® Non-Stick RNase-free Microfuge Tubes (0.5 mL), 500 AM12350
Ambion® Non-Stick RNase-free Microfuge Tubes (1.5 mL), 250 AM12450
Ambion® Nuclease-free Water (not DEPC-treated), 500 mL AM9930
Ethanol, 100%, ACS reagent grade or equivalent MLS
Pipette tips, RNase-free MLS

For whole transcriptome libraries: standard input

Item Catalog no.

Ambion® ATP Soln (optional) AM8110G
Ambion® T4 Polynucleotide Kinase (Cloned) 10 U/µL (optional) AM2310
Invitrogen™ Qubit® 2.0 Fluorometer Q32866
Centrifugal vacuum concentrator (for example, SpeedVac) MLS
Invitrogen™ PureLink® PCR Micro Kit, 50 preps K310050
Invitrogen™ Quant-iT™ RNA Assay Kit, 100 assays Q32852

10 SOLiD® Total RNA-Seq Kit

 Chapter 1 SOLiD™ Total RNA-Seq Kit
Product information 1

For whole transcriptome libraries: standard input

Item Catalog no.

Invitrogen™ RiboMinus™ Concentration Module, 6 preps K1550-05
Note: The RiboMinus™ Concentration Module is not equivalent to the
RiboMinus™ Eukaryote Kit for RNA-Seq or to the RiboMinus™ Plant
Kit for RNA-Seq.
Agilent® DNA 1000 Kit Agilent
5067-1504
Agilent® RNA 6000 Pico Kit Agilent
5067-1513
Agencourt® AMPure® XP, 5mL Beckman Coulter
Genomics A63880
Ambion® Magnetic Stand-96 AM10027
OR
AM10050
U-bottom microplates, 96-well MLS
Ambion MicroPoly(A)Purist™ Kit (optional) AM1919
Invitrogen RiboMinus™ Eukaryote Kit for RNA-Seq (optional) A10837
Invitrogen RiboMinus™ Plant Kit for RNA-Seq (optional) A10838

For whole transcriptome libraries: low input

Item Catalog no.

Ambion® Magnetic Stand-96 AM10027
Invitrogen™ PureLink® RNA Micro Kit 12183-016
Agencourt® AMPure® XP, 5mL Beckman Coulter
Genomics A63880
Agilent® DNA 1000 Kit Agilent
5067-1504
Agilent® High Sensitivity DNA Kit (optional) Agilent
5067-4626
Agilent® RNA 6000 Pico Kit Agilent
5067-1513
U-bottom microplates, 96-well MLS
mRNA Catcher™PLUS Kit (optional) Invitrogen
K157002
Ambion® MicroPoly(A)Purist™ Kit (optional) AM1919

SOLiD® Total RNA-Seq Kit 11

 Chapter 1 SOLiD™ Total RNA-Seq Kit
1 Product information

For small RNA libraries

Item Catalog no.

Invitrogen™ XCell SureLock™ Mini-Cell EI0001
Transilluminator MLS
Invitrogen™ 10 bp DNA Ladder 10821-015
Invitrogen™ Novex® 10% TBE-Urea Gels 1.0 mm, 10 well EC6875BOX
Invitrogen™ Novex® TBE-Urea Sample Buffer (2✕), 10 mL LC6876
Invitrogen™ Novex® TBE Running Buffer (5✕), 1 L LC6675
Invitrogen™ PureLink® PCR Micro Kit, 50 preps K310050
Invitrogen™ SYBR® Gold nucleic acid gel stain, 10,000✕ concentrate S-11494
in DMSO, 500 µL
Agilent® High-Sensitivity DNA Kit (optional) Agilent
5067-4626
Agilent® DNA 1000 Kit Agilent
5067-1504
Agilent® RNA 6000 Nano Kit Agilent
5067-1511
Agilent® Small RNA Kit Agilent
5067-1548
MinElute® PCR Purification Kit (50) Qiagen
28004

12 SOLiD® Total RNA-Seq Kit

 2 Whole Transcriptome Library
Preparation: Standard Input

Fragment the whole transcriptome RNA (standard input)

Start with 100–500 ng poly(A) RNA or 200–500 ng rRNA-depleted total RNA
(We strongly recommend that you add ERCC RNA Spike-In Control Mixes to the
input total RNA see page 14)

Fragment the RNA:

Fragment the RNA using RNase III (page 15) OR
Fragment the RNA by chemical hydrolysis (page 16) Fragmented RNA

Clean up the fragmented RNA (page 17)

Assess the yield and size distribution of the fragmented RNA (page 18)

Construct the amplified whole transcriptome library (standard input)

Hybridize and ligate the RNA (page 21)

Perform reverse transcription (page 22)

Purify and size-select the cDNA: round 1 (page 23)

Purify and size-select the cDNA: round 2 (page 24)

5′ PCR Primer

(Barcode)
Amplify the cDNA (page 25) optional

3′ PCR Primer

P1 sequence internal adaptor (IA) P2 sequence

Purify the amplified DNA (page 27) RNA sequence barcode (BC)

Assess the yield and size distribution of the amplified DNA (page 28)

Proceed with SOLiD™ System templated bead preparation

Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead Preparation P1
Guide (PN 4448378) or to IA BC
P2
SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no. 4441486)

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Guidelines for RNA sample type and amount

Guidelines for RNA sample type and amount

We highly recommend the addition of Ambion® ERCC Spike-In Control Mixes (Part
nos. 4456739 and 4456739) to the input RNA at the earliest step possible, preferably at
the total RNA stage following the guidelines below, prior to whole transcriptome
library preparation.
• The Ambion® ERCC RNA Spike-In Control Mixes provide a set of external RNA
controls that enable performance assessment of a variety of technology platforms
used for gene expression experiments. Add one Spike-In Mix to each RNA
sample, and run the Spike-In Mix-containing samples on your platform. Then
compare the Spike-In Mix data to known Spike-In Mix concentrations and ratios
to assess the dynamic range, lower limit of detection, and fold-change response of
your platform.
• The Ambion® ERCC RNA Spike-In Control Mixes are pre-formulated sets of 92
polyadenylated transcripts from the ERCC plasmid reference library. The
transcripts are traceable through the manufacturing process to the NIST plasmid
reference material. For detailed information, please refer to the ERCC Spike-In
Control protocol. For your convenience, we have included the following table as
reference during experimental design.
Guidelines for adding Spike-In Mixes to sample RNA

Volume of Spike-In Mix 1 or Mix 2 (dilution]†

Amount of sample
RNA
Total RNA Poly(A) RNA

20 ng 4 µL (1:10000) 2 µL (1:100)
50 ng 1 µL (1:1000) 5 µL (1:100)
100 ng 2 µL (1:1000) 1 µL (1:10)
500 ng 1 µL (1:100) 5 µL (1:10)
1000 ng 2 µL (1:100)
5000 ng 1 µL (1:10)
† ERCC RNA Spike-In Mix 1, ExFold Spike-In Mix 1, or ExFold Spike-In Mix 2.

Use highest-quality RNA available as your starting material. Ambion® FirstChoice®

Total RNA provides high-quality, intact RNA isolated from a variety of sources. Best
results are obtained when using RNA with an RNA integrity number (RIN) greater
than 7.
Use 100–500 ng poly(A) RNA or 200–500 ng rRNA-depleted total RNA.
• For poly(A) RNA, we recommend performing two rounds of oligo(dT) selection
of the poly(A) RNA; for example, use the Ambion MicroPoly(A)Purist™ Kit. Also,
confirm the absence of 18S and 28S rRNA; for example, check the profile of the
poly(A) RNA on an Agilent® 2100 Bioanalyzer™ Instrument.
• For rRNA-depleted total RNA, we recommend that you remove rRNA from total
RNA for transcriptome analysis using the Invitrogen RiboMinus™ Eukaryote Kit
for RNA-Seq or the Invitrogen RiboMinus™ Plant Kit for RNA-Seq. Also, confirm
the absence of 18S and 28S rRNA; for example, check the profile of the rRNA-
depleted RNA on an Agilent® 2100 Bioanalyzer™ Instrument.

14 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Fragment the whole transcriptome RNA 2

Fragment the whole transcriptome RNA

In this guide, we provide procedures for two methods of RNA fragmentation,
enzymatic and chemically induced. Each fragmentation method has pros and cons.
Both fragmentation methods have been verified as part of the library preparation
procedure outlined in this protocol. For more information and to help you select the
best RNA fragmentation method for your experiment, see , “About the RNA
fragmentation methods” on page 73. Use information about your experimental goals
along with this information to choose the optimal fragmentation method for your
study.
Fragmentation of the whole transcriptome RNA involves the following procedures:

1. Fragment the RNA:

• Fragment the RNA using RNase III (page 15)
OR
• Fragment the RNA by chemical hydrolysis (page 16)

2. Clean up the fragmented RNA (page 17)

3. Assess the yield and size distribution of the fragmented RNA (page 18)

Fragment the RNA Use components from the SOLiD™ Total RNA-Seq Kit:
using RNase III • Nuclease-free Water
• 10✕ RNase III Reaction Buffer
• RNase III

1. For each RNA sample, assemble the reaction on ice:

Component (add in order shown) Volume

RNA sample and Nuclease-free Water: 8 µL

• Poly(A) RNA: 100–500 ng
• rRNA-depleted total RNA: 200–500 ng
• WT Control RNA: 500 ng
10✕ RNase III Reaction Buffer 1 µL
RNase III 1 µL
Total volume 10 µL

IMPORTANT! To reduce fragmentation variability, accurately pipet 1 µL of 10✕

RNase III Reaction Buffer and 1 µL of RNase III to each sample. Do not make a
master mix that contains only 10✕ RNase III Reaction Buffer and RNase III.

2. Flick the tube or pipet up and down a few times to mix, then spin briefly.
3. Incubate the reaction in a thermal cycler at 37°C for 10 minutes.
4. Immediately after the incubation, add 90 µL of Nuclease-free Water, then place the
fragmented RNA on ice. Proceed with cleaning up the fragmented RNA
immediately (page 17), or leave the fragmented RNA on ice for less than 1 hour.

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Fragment the whole transcriptome RNA

Fragment the RNA Note: If you have fragmented the RNA using the RNase III procedure, skip this section
by chemical and proceed to, “Clean up the fragmented RNA” on page 17.
hydrolysis For the RNA fragmentation, use components from the SOLiD™ Total RNA-Seq Kit:
• Nuclease-free Water
• 10✕ RNase III Reaction Buffer
For the kinase reaction, use the following components not included in the SOLiD Total
RNA-Seq Kit:
• Ambion® T4 Polynucleotide Kinase (Cloned) 10 U/µL
• Ambion® ATP Soln.

1. For each RNA sample, assemble the reaction on ice:

Component (add in order shown) Volume

RNA sample and Nuclease-free Water: 9 µL

• Poly(A) RNA: 100–500 ng
• rRNA-depleted total RNA: 200–500 ng
• WT Control RNA: 500 ng
10✕ RNase III Reaction Buffer 1 µL
Total volume 10 µL

2. Flick the tube or pipet up and down a few times to mix, then spin briefly.
3. Incubate the reaction in a thermal cycler at 95°C for 10 minutes.

IMPORTANT! Incubate exactly 10 minutes. Shortening or lengthening the

incubation time can result in suboptimal size distribution of the fragmented RNA.

4. Immediately after the incubation, snap-cool the tube on ice.

IMPORTANT! Proceed with the next step immediately.

5. To each 10 µL fragmented RNA sample, add the kinase reaction components:

Component (add in order shown) Volume

Ambion® T4 Polynucleotide Kinase (Cloned) 10 U/μL 1 µL

Ambion® ATP Soln 1 µL

6. Flick the tube or pipet up and down a few times to mix, then spin briefly.
7. Incubate the reaction in a thermal cycler at 37°C for 30 minutes.
8. Immediately after the incubation, add 88 µL of Nuclease-free Water, then place the
fragmented RNA on ice.

Proceed with cleaning up the fragmented RNA immediately (page 17), or leave the
fragmented RNA on ice for less than 1 hour.

16 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Fragment the whole transcriptome RNA 2

Clean up the Use the Invitrogen RiboMinus™ Concentration Module.

fragmented RNA Note: Alternatively, use the Purelink® RNA Micro Kit, following the instructions on
page 37. If you follow the alternative clean-up procedure, make sure that you return to
the procedures in this chapter for constructing the amplified whole transcriptome
library.

1. Add Binding Buffer (L3) and ethanol to the fragmented RNA, then mix well:
• 100 µL of Binding Buffer (L3)
• 250 µL of 100% ethanol

2. Bind the RNA sample containing Binding Buffer (L3) and ethanol to the Spin
Column:
a. Place the Spin Column in a clean 1.5-mL Wash Tube.
b. Load 450 µL of the RNA sample containing Binding Buffer (L3) and ethanol
onto the Spin Column.
c. Spin the column at 12,000 × g for 1 minute.
d. Discard the flowthrough.

3. Prepare the Wash Buffer (W5) with ethanol, then store at room temperature:

Component Volume

100% ethanol 6 mL
Wash Buffer (W5) 1.5 mL

4. Wash the RNA:

a. Return the Spin Column to the Wash Tube.
b. Add 500 µL of Wash Buffer (W5) with ethanol to the Spin Column.
c. Spin the column at 12,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the Spin Column in the Wash Tube.
f. Spin the column at maximum speed for 2 minutes.

5. Elute the RNA in a clean Recovery Tube:

a. Place the Spin Column in a clean Recovery Tube.
b. Add 12 µL of Nuclease-Free water to the center of the Spin Column.

IMPORTANT! Use the RNase-Free water from your SOLiD Total RNA-Seq
Kit. Other nuclease-free water may contain DEPC which will affect
downstream enzymatic reactions.

c. Wait 1 minute, then spin the column at maximum speed for 1 minute.

Note: You should recover approximately 10 µL of fragmented RNA from the

column.

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Fragment the whole transcriptome RNA

Assess the yield Use the Invitrogen Quant-iT™ RNA Assay Kit with the Qubit® Fluorometer and the
and size Agilent® RNA 6000 Pico Chip Kit with the Agilent® 2100 Bioanalyzer™ Instrument.
distribution of the Note: You can use a NanoDrop® Spectrophotometer in place of the Quant-iT RNA
fragmented RNA Assay Kit and Qubit Fluorometer. However, RNA eluted from spin columns may
contain extra salts or other components that affect readings on the NanoDrop®
Spectrophotometer. For increased accuracy, quantitate the RNA concentration using
the Quant-iT RNA Assay Kit on the Qubit Fluorometer.

1. Quantitate the yield of the fragmented RNA using the Quant-iT RNA Assay Kit
on the Qubit Fluorometer.
Refer to the Quant-iT™ RNA Assay Kit Protocol or the Qubit® Fluorometer
Instruction Manual for instructions.

2. Assess the size distribution of the fragmented RNA:

a. Dilute 1 µL of the sample 1:10 with Nuclease-Free Water.
b. Run the diluted sample on an Agilent® 2100 Bioanalyzer™ Instrument with
the RNA 6000 Pico Chip Kit. Follow the manufacturer’s instructions for
performing the assay.
c. Using the 2100 expert software, review the size distribution.
The fragmentation procedure should produce a distribution of RNA
fragment sizes from 35 nt to several hundred or a few thousand nt,
depending on your sample type. The median size is normally 125–200 nt. See
Figures 1-5 starting on page 19.
Note: For instructions on how to review the size distribution, refer to the
Agilent® 2100 Bioanalyzer™ 2100 Expert User’s Guide by Agilent®.
If the profile for the fragmented RNA does not meet the typical results, see
“Troubleshooting” on page 31 for guidance.

3. Proceed according to the amount of fragmented RNA you have in 3 µL:

Amount of fragmented
Instructions
RNA in 3 µL

• ≥50 ng poly(A) RNA Proceed with “Construct the amplified whole

• ≥100 ng rRNA- transcriptome library” on page 20.
depleted total RNA Store the remaining RNA at –80°C.
• ≥100 ng WT Control
RNA
• <50 ng poly(A) RNA 1. Dry 50–100 ng of the RNA completely in a
• <100 ng rRNA- centrifugal vacuum concentrator at low or medium
heat (≤40°C); this should take 10–20 minutes.
depleted total RNA
2. Resuspend in 3 µL Nuclease-free Water, then
proceed with “Construct the amplified whole
transcriptome library” on page 20.

18 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Fragment the whole transcriptome RNA 2

Typical results of Figures 1-3 show profiles from an Agilent® 2100 Bioanalyzer™ Instrument after RNase
fragmentation of III fragmentation and cleanup. Figure 1 shows results with HeLa poly(A) RNA.
Figure 2 shows results with rRNA-depleted HeLa RNA. Figure 3 shows results with
whole
WT control total RNA. Figures 4-5 show profiles from an Agilent 2100 Bioanalyzer
transcriptome RNA Instrument after chemical hydrolysis and cleanup. Figure 4 shows results with Hela
poly(A) RNA. Figure 5 shows results with rRNA-depleted Hela RNA.
Figure 1 Size distribution of RNAse III fragmented HeLa poly(A) RNA (median size is 165 nt)

Figure 2 Size distribution of RNAse III fragmented rRNA-depleted HeLa RNA (median size is
140 nt)

Figure 3 Size distribution of RNAse III fragmented WT Control total RNA (median size is 195 nt)

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Construct the amplified whole transcriptome library

Figure 4 Size distribution of chemical fragmented Hela poly(A) RNA (median size is 165 nt)

Figure 5 Size dist. of chemical fragmented rRNA-depleted Hela RNA (median size is 138 nt)

Construct the amplified whole transcriptome library

Constructing the amplified whole transcriptome library involves the following
procedures:

1. Hybridize and ligate the RNA (page 21)

2. Perform reverse transcription (page 22)
3. Purify and size-select the cDNA: round 1 (page 23)
4. Purify and size-select the cDNA: round 2 (page 24)
5. Amplify the cDNA (page 25)
6. Purify the amplified DNA (page 27)
7. Assess the yield and size distribution of the amplified DNA (page 28)
8. Proceed with SOLiD™ System templated bead preparation (page 29)

20 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Construct the amplified whole transcriptome library 2

Hybridize and Use components from the SOLiD™ Total RNA-Seq Kit:
ligate the RNA • SOLiD™ Adaptor Mix
• Hybridization Solution
• Nuclease-free Water
• 2✕ Ligation Buffer
• Ligation Enzyme Mix

1. On ice, prepare the hybridization master mix:

Volume
Component for one
reaction†

SOLiD™ Adaptor Mix 2 µL

Hybridization Solution 3 µL
Total volume per reaction 5 µL
† Include 5–10% excess volume to compensate for pipetting error.

2. Transfer 5 µL hybridization master mix to 3 µL fragmented RNA sample:

• Fragmented poly(A) RNA: 50 ng
• Fragmented rRNA-depleted total RNA: 100 ng
• Fragmented WT Control RNA: 100 ng

3. Slowly pipet up and down a few times to mix well, then spin briefly.
4. Run the hybridization reaction in a thermal cycler:

Temperature Time

65°C 10 min
16°C 5 min

5. Add the RNA ligation reagents to each 8-µL hybridization reaction:

Component (add in order shown) Volume

2✕ Ligation Buffer 10 µL
Ligation Enzyme Mix 2 µL

IMPORTANT! You may observe a white precipitate in the 2✕ Ligation Buffer. If so,
warm the tube at 37°C for 2–5 minutes or until the precipitate is dissolved.
2✕ Ligation Buffer is very viscous; pipet slowly to dispense it accurately.

6. Flick the tube or slowly pipet up and down a few times to mix well, then spin
briefly.

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Construct the amplified whole transcriptome library

7. Incubate the 20-µL ligation reaction in a thermal cycler at 16°C for 16 hours.
Note: If possible, set the temperature of the thermal cycler lid to match the block
temperature. Otherwise, incubate the reaction with the heated lid turned off, or
do not cover the reaction tubes with the heated lid.

Perform reverse Use components from the SOLiD™ Total RNA-Seq Kit:
transcription • Nuclease-free Water
• 10✕ RT Buffer
• dNTP Mix
• SOLiD™ RT Primer
• ArrayScript™ Reverse Transcriptase

1. Prepare RT master mix (without the ArrayScript™ Reverse Transcriptase):

Volume for
Component one
reaction†

Nuclease-free Water 11 µL
10X RT Buffer 4 µL
dNTP Mix 2 µL
SOLiD™ RT Primer 2 µL
Total volume per reaction 19 µL
† Include 5–10% excess volume in the master mix to compensate for pipetting error.

2. Incubate the RT master mix with the ligated RNA sample:

a. Add 19 µL of RT master mix to each 20-µL ligation reaction.
b. Pipet up and down a few times to mix, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 70°C for 5 minutes, then
snap-cool on ice.

3. Perform the reverse transcription reaction:

a. Add 1 µL ArrayScript™ Reverse Transcriptase to each ligated RNA sample.
b. Gently vortex to mix thoroughly, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 42°C for 30 minutes.

Note: The cDNA can be stored at –20°C for a few weeks, stored at –80°C for long-
term storage, or used immediately.

22 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Construct the amplified whole transcriptome library 2

Purify and size- Using Agencourt® AMPure® XP Reagent, perform two rounds of bead capture, wash,
select the cDNA: and elution to ensure complete capture and size-selection of the desired cDNA.
round 1 Note: If you require cDNA libraries with more accurate, defined insert sizes, you may
wish to use the gel-based size selection protocol on page 74.

The first round of bead capture removes the salt and enzyme and binds cDNA
products greater than 100 bp with a 1.8✕ addition of bead volume to the sample
mixture. The subsequent washes remove fragments that are less than 100 bp. This
process increases the percentage of library inserts that are in the desired size range.
The protocol described below uses an U-bottom 96 well plate and single or multi-
channel pipets for mixing steps. Alternatively, a 1.2ml 96-Well plate and a plate shaker
could be used assuming the beads are homogeneously resuspended at each mixing
step.
Required materials:
• Agencourt® AMPure® XP Beads (place at room temperature 30 minutes before
using)
• U-bottom microplate
• Magnetic Stand-96
• 70% ethanol, freshly prepared from 100% ethanol, ACS reagent grade or
equivalent and nuclease-free water
• Nuclease-free Water

1. Bind the cDNA to the beads:

a. Gently shake the Agencourt® AMPure® XP bottle to resuspend any magnetic
particles that may have settled.
b. Add 72 µL Agencourt® AMPure® XP Reagent to each 40 µL sample. Pipet
up and down a few times to mix.
c. Transfer the 112-µL bead and sample mixture to a U-bottom microplate.
d. Incubate samples for 5 minutes at room temperature.

2. Remove the buffer from the beads:

a. Place a U-bottom plate onto a Magnetic Stand-96 for 2-5 minutes to separate
beads from the solution. Wait for the solution to clear before proceeding to
the next step.
b. While the plate is in the magnetic stand, aspirate the supernatant from the
plate and discard.

IMPORTANT! This step must be performed while the reaction plate is on the
magnetic stand. Do not disturb the beads. If necessary, leave a few
microliters of supernatant rather than disturb the beads.

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Construct the amplified whole transcriptome library

3. Wash the beads with 70% ethanol:

a. While the plate is on the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the U-bottom plate and incubate for 30 seconds at room
temperature. Then aspirate the ethanol and discard.

IMPORTANT! This step must be performed while the reaction plate is on the
magnetic stand. Do not disturb the beads.

b. Remove the plate from the Magnetic Stand-96.

4. Elute the cDNA from the beads:

a. Add 40 µL of Nuclease-free water to each well of the plate.
Note: Elution occurs quickly. It is not necessary for the beads to go back into
solution for it to occur.
b. Place U-bottom plate onto a Magnetic Stand-96 for 2-5 minutes to separate
beads from the solution. Wait for the solution to clear before proceeding to
the next step.
c. Transfer 40 µL eluant to an empty well of the U-bottom plate for the second
round of purification and size-selection. Then remove plate from Magnetic
Stand-96.
Note: If processing is equal to or less than 40 samples at one time, you may
use the same U-bottom plate for both rounds of purification and size-
selection. To avoid contamination, leave an empty row of wells between the
two rounds.

Purify and size- The second round of bead capture using Agencourt® AMPure® XP binds cDNA
select the cDNA: products greater than 150 bp with a 1.6✕ addition of bead volume to the sample
mixture. Two rounds of size selection are required to increase the percentage of library
round 2
inserts that are in the desired size range.

1. Bind the cDNA from the first bead capture to the beads:
a. Gently shake the Agencourt® AMPure® XP Reagent bottle to resuspend any
magnetic particles that may have settled.
b. Add 64 µL Agencourt® AMPure® XP Reagent to each 40 µL of sample from
the first bead capture.
c. Mix thoroughly by pipette mixing 10 times. The color of the mixture should
appear homogenous after mixing.
d. Incubate samples for 5 minutes at room temperature to bind cDNA products
to the magnetic beads.

24 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Construct the amplified whole transcriptome library 2

2. Remove the buffer from the beads:

a. Place U-bottom plate on a Magnetic Stand-96 for 2-5 minutes to separate
beads from the solution. Wait for the solution to clear before proceeding to
the next step.
b. While the plate is in the magnetic stand, aspirate the supernatant from the
plate and discard.

IMPORTANT! This step must be performed while the reaction plate is on the
magnetic stand. Do not disturb the beads. If necessary, leave a few
microliters of supernatant rather than disturb the beads.

3. Wash the beads with 70% ethanol:

a. While the plate is in the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the U-bottom plate and incubate for 30 seconds at room
temperature.
b. Aspirate the cleared solution from the reaction plate and discard.

IMPORTANT! Perform these steps with U-bottom plate situated on the

Magnetic Stand-96. Do not disturb the separated magnetic beads. Be sure to
remove all of the ethanol from the bottom of the well as it is a known PCR
inhibitor.

c. Off the Magnetic Stand-96, allow the beads to dry for 5 minutes at room
temperature.
Note: Overdrying beads significantly decreases elution efficiency, however,
leftover ethanol may inhibit PCR.

4. Elute the cDNA:

a. While the plate is off the magnetic stand, add 40 µL of Nuclease-free water to
each well of the U-bottom plate and pipette mix 10 times.
Note: Elution occurs quickly. It is not necessary for the beads to go
completely back into solution for elution to occur. Some beads may appear
clumpy.
b. Place U-bottom plate on a Magnetic Stand-96 for 2-5 minutes to separate
beads from the solution. Wait for the solution to clear before proceeding to
the next step.
c. Transfer the eluant (40 µL) to a new 0.2mL tube or plate.

Amplify the cDNA Use components from the SOLiD™ Total RNA-Seq Kit:
• Nuclease-free water
• 10x PCR buffer
• dNTP Mix
• SOLiD™ 5’ PCR Primer
• AmpliTaq® DNA Polymerase
• SOLiD™ 3’ PCR Primer

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 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
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(Optional) To prepare cDNA libraries for multiplex SOLiD System sequencing,

substitute with the barcoded SOLiD™ 3′ Primers from one of the SOLiD™ RNA
Barcoding Kits.
Note: Plan your experiments to include multiples of four different barcoded libraries
in every multiplex sequencing pool, to preserve color balance on the SOLiD System
sequencing run. Use the SOLiD 3′ Primers from the SOLiD RNA Barcoding Kit in the
appropriate color-balanced groups. For more information, refer to the product insert
for your SOLiD RNA Barcoding Kit and the Applied Biosystems SOLiD™ 4 System SETS
Software User Guide (PN 4448411).

1. For each cDNA sample, prepare 45 µL PCR mix.

Component Volume (µL)

50 µL PCR reaction

Nuclease-free water 33.4

10x PCR Buffer 5.0
dNTP Buffer 4.0
SOLiD 5’ PCR Primer 1.0
AmpliTaq® DNA Polymerase 0.6
Total Volume 44.0

2. Transfer 5 µL of each cDNA sample into a new 0.2mL PCR tube.

3. Add 44 µL PCR mix to each cDNA sample.
4. Add 1 µL 3' PCR Primer to each sample.
Note: To prepare cDNA libraries for multiplex SOLiD™ System sequencing,
substitute with the barcoded SOLiD™ 3' PCR Primers from one of the SOLiD™
RNA Barcoding Kits.

5. Run the PCR reactions in a thermal cycler:

Stage Temperature Time

Hold 95°C 5 minutes

95°C 30 seconds
Cycle (15 cycles) 62°C 30 seconds
72°C 30 seconds
Hold 72°C 7 minutes

Note: Run 15 cycles if you started with 50-100ng of fragmented RNA. If

necessary, increase the volume of cDNA used for each PCR reaction, or adjust the
numbers of cycles according to the amount of fragmented RNA used in the
hybridization and ligation steps, but for optimal results run between 12 and 18
cycles. Too many cycles results in overamplification and changes the expression
profile.

26 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Construct the amplified whole transcriptome library 2

Purify the amplified DNA

Use the Invitrogen PureLink® PCR Micro Kit.
• PureLink® Micro Kit Column
• Collection Tube
• Binding Buffer (B2)
• Wash Buffer (W1)
• PureLink® Elution Tube

IMPORTANT! Do not use other PCR purification kits. Other purification kits are not as
effective in the removal of unincorporated primers. Unincorporated primers can affect
the final quantitation and emulsion PCR.

1. Before using the PureLink® Micro Kit Column, place it in a clean Collection Tube,
then spin the column at 10,000 × g for 1 minute.

2. Prepare the sample:

a. Transfer the 50-µL PCR product into a new 1.5-mL tube.

IMPORTANT! If you used two different barcoded SOLiD 3′ Primers for your
sample, do not combine the PCRs at this step.

b. Add 200 µL of Binding Buffer (B2) to the tube, then mix well.

3. Load the sample onto the PureLink® Micro Kit Column:

a. Load 250 µL of the sample containing Binding Buffer (B2) onto the
PureLink® Micro Kit Column.
b. Spin the column at 10,000 × g for 1 minute.
c. Discard the flowthrough.

4. Wash the DNA:

a. Return the column to the Collection Tube.
b. Add 600 µL of Wash Buffer (W1) to the column.
c. Spin the column at 10,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the column to the Collection Tube.
f. Spin the column at 14,000 × g for 1 minute.

5. Elute the DNA in a clean PureLink® Elution Tube:

a. Place the column in a clean PureLink® Elution Tube.
b. Add 10 µL of Elution Buffer to the center of the membrane.
c. Wait 1 minute, then spin the column at 14,000 × g for 1 minute.
d. Repeat step 5b through step 5c for a total elution volume of 20 µL.

SOLiD® Total RNA-Seq Kit 27

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Construct the amplified whole transcriptome library

Assess the yield Use a NanoDrop® Spectrophotometer, and the Agilent® 2100 Bioanalyzer™
and size Instrument with the DNA 1000 Kit (Agilent®). For instructions on how to perform a
smear analysis, see page 88.
distribution of the
amplified DNA 1. Measure the concentration of the purified DNA with a NanoDrop®
Spectrophotometer, and if necessary, dilute the DNA to <50 ng/µL for accurate
quantitation with the DNA 1000 Kit.

2. Run 1 µL of the purified DNA on an Agilent® 2100 Bioanalyzer™ Instrument with

the DNA 1000 Kit. Follow the manufacturer’s instructions for performing the
assay.

3. Using the 2100 expert software, perform a smear analysis to quantify the
percentage of DNA that is 25–200 bp.
Note: In general, we see a positive correlation between RNA-Seq mapping
statistics and the percent of DNA library inserts that are above 200 bp in length.
We recommend minimizing the percent of inserts that fall in the 25-200 bp range.
Please use your judgement when deciding the best way to proceed with libraries
from precious samples that fall close to these recommended quality values of
more than 50% DNA in 25-200 bp range.

Percent of DNA in
Next steps
the 25–200 bp range

Less than 50% Proceed with SOLiD™ System templated bead

preparation.
50-60% You may perform another round of purification and size-
selection on the amplified DNA using Agencourt®
AMPure® XP:
1. Bring the sample volume to 40 µL with Nuclease-free
Water.
2. Follow the instructions in “Purify and size-select the
cDNA: round 2” on page 24, using AMPureXP bead
purification to decrease the % of amplified DNA in the
25-200bp range. However, in order to maintain a high
concentration, elute with 20 µL of water, instead of
with the 40 µL used in step 4 of the procedure.
OR
Alternatively, you may proceed with SOLiD System
templated bead preparation and expect to see slightly
higher % of filtered reads in your sequencing data
when compared to libraries with less than 50% in the
25-200bp range.
More than 60% It is recommended that you perform another round of
purification and size-selection using Agencourt
AMPureXP. See step 1 and step 2 above.

Note: For instructions on how to perform the smear analysis, see “Perform a
smear analysis” on page 88. You may also refer to the Agilent® 2100 Bioanalyzer™
2100 Expert User’s Guide by Agilent®.

28 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Construct the amplified whole transcriptome library 2

4. Determine the median peak size (bp) and molar concentration (nM) of the cDNA
library using the Agilent® software. For more information, see “Determine the
median size” on page 89.
Note: The mass concentration of the cDNA must be <50 ng/µL for accurate
quantitation with the DNA 1000 Kit.

Alternatively, obtain the mass concentration by another method, and convert the
mass concentration to molar concentration.

Proceed with When the amplified DNA in the 25–200 bp range is minimal, you can proceed with the
SOLiD™ System SOLiD™ System templated bead preparation stage, in which each library template is
clonally amplified on SOLiD™ P1 DNA Beads by emulsion PCR. Refer to the Applied
templated bead
Biosystems SOLiD™ 4 System Templated Bead Preparation Guide (PN 4448378) or to
preparation SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no. 4441486).
Barcoded libraries are pooled prior to templated bead preparation. For more
information refer to the product insert for your SOLiD™ RNA Barcoding Kit.
Note: When optimizing SOLiD Total RNA-Seq Kit library concentrations (singleplex
or multiplex sequencing pools) by workflow analysis (WFA), library concentrations of
0.4 pM and 0.8 pM for ePCR are recommended.

Typical size profiles Typical size distributions (Agilent® 2100 Bioanalyzer™ Instrument profiles) of
of amplified amplified libraries prepared from HeLa poly(A) RNA (Figure 6), HeLa WT Control
total RNA (Figure 7), and rRNA-depleted HeLa RNA (Figure 8) using the SOLiD™
libraries
Total RNA-Seq Kit are shown.
Figure 6 Size distribution of amplified library prepared from HeLa poly(A) RNA

SOLiD® Total RNA-Seq Kit 29

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Construct the amplified whole transcriptome library

Figure 7 Size distribution of amplified library prepared from WT control total RNA

Figure 8 Size distribution of amplified library prepared from rRNA-depleted HeLa total RNA

Expected yields The recovery of your experimental RNA will depend on its source and quality. The
following results are typically seen with Human Brain Reference and HeLa RNAs.

Workflow Input amount Typical recovery amount

Fragment the whole transcriptome 500 ng poly(A) RNA, total RNA, or 300–400 ng RNA
RNA (page 15) rRNA-depleted total RNA
Construct the amplified whole 50–100 ng fragmented RNA >100 ng cDNA
transcriptome library (page 20)

30 SOLiD® Total RNA-Seq Kit

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
Troubleshooting 2

Troubleshooting

Observation Possible cause Solution

Agilent® software doesn’t calculate The software detects multiple peaks in Refer to “Analyze multiple peaks as
one concentration and peak size the amplified cDNA profile one peak” on page 90.
Low yield and poor size distribution in You recovered <20% of the input RNA Decrease the RNase III digestion from
the amplified library after you fragmented and cleaned up 10 minutes to 5 minutes (step 3 on
the RNA page 15).
Low yield in the amplified library and RNA fragmentation failed Purify the RNA sample again to
very few differences in the Agilent® remove the extra salts that may affect
2100 Bioanalyzer™ Instrument traces the RNase III activity. If RNA
before and after you fragment the RNA fragmentation still fails, increase the
RNase III digestion from 10 minutes to
20 minutes (step 3 on page 15). For
chemical fragmentation procedure,
slowly increase the fragmentation
time to 12-15 minutes.
Low yield and no PCR products Too much ethanol introduced into PCR Use optional drying step in bead clean
reaction. up protocol.
An enzymatic reaction or column 1. Dilute the cDNA 1:10, then use 1 µL
purification performed after RNase III in a 100-µL PCR.
treatment failed 2. Check the yield before and after
purification using the PureLink®
PCR Micro Kit.
3. If you get the same results, repeat
the ligation with more fragmented
RNA, and run a parallel ligation
reaction with fragmented Control
RNA.
Normal or high yield but PCR products Too many PCR cycles resulted in Decrease the number of PCR cycles.
larger than 300 bp overamplification (step 5 on page 26).

Using a positive A general troubleshooting strategy is to perform the SOLiD™ Total RNA-Seq Kit
control procedure using the WT Control RNA (HeLa total RNA) provided with the kit.
• Use 500 ng WT Control RNA for the fragmentation procedure starting on page 15.
• Use 100 ng fragmented WT Control RNA in the amplified library construction
procedure starting on page 20.
See the expected yields for the WT Control RNA on page 30.

SOLiD® Total RNA-Seq Kit 31

 Chapter 2 Whole Transcriptome Library Preparation: Standard Input
2 Troubleshooting

32 SOLiD® Total RNA-Seq Kit

 3 Whole Transcriptome Library
Preparation: Low Input

Fragment the low input whole transcriptome RNA (low input)

Start with 5-25 ng high quality poly(A) RNA
(We strongly recommend that you add ERCC RNA Spike-In Control Mixes to the
input total RNA see page 34)

Fragment the RNA using RNase III (page 36) Fragmented RNA

Clean up the fragmented RNA (page 37)

Assess the yield and size distribution of the fragmented RNA (page 38)

Construct the amplified low-input whole transcriptome library (low input)

Hybridize and ligate the low input poly(A) RNA (page 39) + NNNNN B NNNNN NNNNN B NNNNN

Perform reverse transcription (page 40)

Purify and size-select the cDNA: round 1 (page 41)

Purify and size-select the cDNA: round 2 (page 42)

5′ PCR Primer

Amplify the cDNA (page 43) (Barcode)

optional

3′ PCR Primer

P1 sequence internal adaptor (IA) P2 sequence

Purify the amplified DNA (page 45)
RNA sequence barcode (BC)

Assess the yield and size distribution of the amplified DNA (page 46)

Proceed with SOLiD™ System templated bead preparation

Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead Preparation P1
Guide (PN 4448378) or to IA BC
P2
SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no. 4441486)

SOLiD® Total RNA-Seq Kit 33

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 About the low input procedures

About the low input procedures

IMPORTANT! Although the low input procedures are similar to the standard
procedures, there are significant differences. For best results, follow these procedures
as written.

We have developed and validated a procedure for preparing whole transcriptome

libraries starting with 5–25 ng of poly(A) RNA. Modifications to the standard input
procedures include:
• Reduced incubation time for RNA fragmentation to produce greater numbers of
RNA fragments within the desired size range
• Modified purification methods after RNA fragmentation to maximize sample
recovery
• Modified PCR conditions to maximize cDNA library yields without introducing a
detectable bias in the expression profile
Note: We do not yet support preparation of whole transcriptome libraries starting
with low inputs of rRNA-depleted total RNA. A protocol for low input rRNA depleted
total RNA is under development. Although this protocol has been optimized for 2-25
ng inputs, we would recommend the use of the low input recommendations for input
amounts from 25-100 ng as well.

We highly recommend the addition of ERCC Spike-In Control Mixes (Part nos. 4456739
and 4456739) to the input RNA at the earliest step possible, preferably at the total RNA
stage following the guidelines below, prior to whole transcriptome library preparation.
• The Ambion®ERCC RNA Spike-In Control Mixes provide a set of external RNA
controls that enable performance assessment of a variety of technology platforms
used for gene expression experiments. Add one Spike-In Mix to each RNA
sample, and run the Spike-In Mix-containing samples on your platform. Then
compare the Spike-In Mix data to known Spike-In Mix concentrations and ratios
to assess the dynamic range, lower limit of detection, and fold-change response of
your platform.
• The ERCC RNA Spike-In Control Mixes are pre-formulated sets of 92
polyadenylated transcripts from the ERCC plasmid reference library. The
transcripts are traceable through the manufacturing process to the NIST plasmid
reference material. For detailed information, please refer to the ERCC Spike-In
Control protocol. For your convenience, we have included the following table as
reference during experimental design.

34 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Fragment the low input whole transcriptome RNA 3

Guidelines for adding Spike-In Mixes to sample RNA

Volume of Spike-In Mix 1 or Mix 2 (dilution]†

Amount of sample
RNA
Total RNA Poly(A) RNA

20 ng 4 µL (1:10000) 2 µL (1:100)
50 ng 1 µL (1:1000) 5 µL (1:100)
100 ng 2 µL (1:1000) 1 µL (1:10)
500 ng 1 µL (1:100) 5 µL (1:10)
1000 ng 2 µL (1:100)
5000 ng 1 µL (1:10)
† ERCC RNA Spike-In Mix 1, ExFold Spike-In Mix 1, or ExFold Spike-In Mix 2.

Use the highest-quality RNA available as your starting material. Ambion®

FirstChoice® Total RNA provides high-quality, intact RNA isolated from a variety of
sources. Best results are obtained when using RNA with an RNA integrity number
(RIN) greater than 7.

Fragment the low input whole transcriptome RNA

Fragmentation of the low input whole transcriptome RNA involves the following
procedures:

1. Fragment the RNA using RNase III (page 36)

2. Clean up the fragmented RNA (page 37)
3. Assess the yield and size distribution of the fragmented RNA (page 38)

Guidelines for RNA Use 5–25 ng of highest-quality poly(A) RNA available as your starting material. For
sample type and example, for best results, use RNA with an RNA integrity number (RIN) greater than
7.
amount
We recommend the following:
• Use the Invitrogen mRNA Catcher™PLUS Kit to isolate mRNA from cells, tissues,
blood, and total RNA.
• Use the Invitrogen mRNA Catcher™ PLUS 96-well plate and reagents for
isolation of polyA-tailed mRNA from small sample sizes of 100ng of Total RNA
up to 100µg of Total RNA.
mRNA Catcher™ PLUS 96-well plate also accommodates polyA-tailed mRNA
selection from 100 cells up to 106 cells per well.
• Alternatively, perform two rounds of oligo(dT) selection of the poly(A) RNA. For
example, use the Ambion® MicroPoly(A)Purist™ Kit which accommodates
2–400 µg total RNA.
• Confirm the absence of 18S and 28S rRNA. For example, check the profile of the
poly(A) RNA on an Agilent® 2100 Bioanalyzer™ Instrument.

SOLiD® Total RNA-Seq Kit 35

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Fragment the low input whole transcriptome RNA

Fragment the RNA Use components from the SOLiD™ Total RNA-Seq Kit:
using RNase III • Nuclease-free Water
• 10✕ RNase III Reaction Buffer
• RNase III

1. For each poly(A) RNA sample, assemble the reaction on ice:

Component (add in order shown) Volume

5–25 ng Poly(A) RNA sample in Nuclease-free Water 8 µL

10✕ RNase III Reaction Buffer 1 µL
RNase III 1 µL
Total volume 10 µL

IMPORTANT! To reduce fragmentation variability, accurately pipet 1 µL of 10✕

RNase III Reaction Buffer and 1 µL of RNase III to each sample. Do not make a
master mix that contains only 10✕ RNase III Reaction Buffer and RNase III.

2. Flick the tube or pipet up and down a few times to mix, then spin briefly.
3. Incubate the reaction in a thermal cycler at 37°C for 3 minutes.

IMPORTANT! The low input protocol only requires 3 minutes of incubation, as

opposed to the 10 minutes required for the standard protocol. Do not over-
incubate.

4. Immediately after the incubation, add 90 µL of Nuclease-free Water, then place the
fragmented low-input RNA on ice.

Proceed with cleaning up the fragmented RNA immediately (page 37), or leave the
fragmented RNA on ice for less than 1 hour.

36 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Fragment the low input whole transcriptome RNA 3

Clean up the Use the Purelink® RNA Micro Kit (Invitrogen Cat no. 12183-016)
fragmented RNA
IMPORTANT! Use of the PureLink® RNA Micro Kit Columns instead of the
RiboMinus™ Concentration Module is strongly recommended for optimum recovery
of low input amounts of RNA.

Note: It is not necessary to add 2-mercaptoethanol or DTT to the Lysis Buffer.

1. Before using Wash Buffer II for the first time:

a. Add 60 mL of 96-100% ethanol directly to the bottle.
b. Check the box on the Wash Buffer II label to indicate that ethanol was added.
c. Store Wash Buffer II with ethanol at room temperature.

2. Transfer the 100 µL fragmented RNA digestion mix to a 1.5mL tube. Add 100 µL
of Lysis Buffer and 250 µL of 100% ethanol, then mix well.

3. Obtain a PureLink® RNA Micro Kit Spin Column with a collection tube, then
verify that the column has a red o-ring.
Note: The red o-ring differentiates the PureLink® RNA Micro Kit Spin Column
from the unmarked PureLink® PCR Micro Kit Spin Column.

4. Bind the RNA to the spin column:

a. Load 450 µL of the RNA sample containing Lysis Buffer and ethanol onto the
Spin Column.
b. Spin the column at 12,000 × g for 15 seconds.
c. Discard the flowthrough.

5. Wash the RNA:

a. Return the Spin Column to the Collection Tube.
b. Add 500 µL of Wash Buffer II (W5) with ethanol to the Spin Column.
c. Spin the column at 12,000 × g for 15 seconds.
d. Discard the flowthrough.

6. Wash the RNA again:

a. Return the Spin Column to the Collection Tube.
b. Add another 500 µL of Wash Buffer II (W5) with ethanol to the Spin Column.
c. Spin the column at 12,000 × g for 15 seconds.
d. Discard the flowthrough.
e. Return the Spin Column to the Collection tube.
f. Using a pipette, carefully remove any buffer adhering to the top of the o-ring
inside of the column.
g. Spin the column at 12,000 × g for 1 minute.

SOLiD® Total RNA-Seq Kit 37

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Fragment the low input whole transcriptome RNA

7. Elute the RNA in a clean Recovery Tube:

a. Place the Spin Column in a clean Recovery Tube.
b. Add 12 µL of Nuclease-free Water to the center of the Spin Column.
c. Wait 1 minute.
d. Spin the column at 12,000 × g for 1 minute.

Note: You should recover approximately 10 µL of fragmented low input RNA

from the column.

Assess the yield Use the RNA 6000 Pico Kit with the Agilent® 2100 Bioanalyzer™ Instrument.
and size 1. Run 1 µL of undiluted sample on an Agilent® 2100 Bioanalyzer™ Instrument with
distribution of the the RNA 6000 Pico Kit. Follow the manufacturer’s instructions for performing the
fragmented RNA assay.

2. Using the 2100 expert software, review the size distribution.

The fragmentation procedure should produce a distribution of RNA fragment
sizes from 35 nt to several hundred or a few thousand nt. The average size should
be 100–200 nt.
Note: For instructions on how to review the size distribution, refer to the Agilent®
2100 Bioanalyzer™ 2100 Expert User’s Guide by Agilent®.

Size distribution of RNAseIII fragmented 5ng input HeLa poly(A) RNA

3. Using a centrifugal vacuum concentrator at low or medium heat (≤40°C) for

approximately 5 - 10 minutes, reduce the sample to 3 µL.

38 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Construct the amplified low-input whole transcriptome library 3

Construct the amplified low-input whole transcriptome library

Constructing the amplified, low-input, whole transcriptome library involves the
following procedures:

1. Hybridize and ligate the low input poly(A) RNA (below)

2. Perform reverse transcription (page 40)
3. Purify and size-select the cDNA: round 1 (page 41)
4. Purify and size-select the cDNA: round 2 (page 42)
5. Amplify the cDNA (page 43)
6. Purify the amplified DNA (page 45)
7. Assess the yield and size distribution of the amplified DNA (page 46)
8. Proceed with SOLiD™ System templated bead preparation (page 47)

Hybridize and Use components from the SOLiD™ Total RNA-Seq Kit:
ligate the low input • SOLiD™ Adaptor Mix
poly(A) RNA • Hybridization Solution
• Nuclease-free Water
• 2✕ Ligation Buffer
• Ligation Enzyme Mix

1. On ice, prepare the hybridization master mix:

Component Volume for one reaction†

SOLiD™ Adaptor Mix 2 µL

Hybridization Solution 3 µL
Total volume per reaction 5 µL
† Include 5–10% excess volume to compensate for pipetting error.

2. Transfer 5 µL hybridization master mix to 3 µL fragmented low input RNA

sample.

3. Slowly pipet up and down a few times to mix well, then spin briefly.
4. Run the hybridization reaction in a thermal cycler:

Temperature Time

65°C 10 min
16°C 5 min

SOLiD® Total RNA-Seq Kit 39

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Construct the amplified low-input whole transcriptome library

5. On ice, prepare the RNA ligation master mix:

Component (add in order shown) Volume

2✕ Ligation Buffer 10 µL
Ligation Enzyme Mix 2 µL
Total volume 12 µL

IMPORTANT! You may observe a white precipitate in the 2✕ Ligation Buffer. If so,
warm the tube at 37°C for 2–5 minutes or until the precipitate is dissolved.
2✕ Ligation Buffer is very viscous; pipet slowly to dispense it accurately.

6. Add 12 µL RNA ligation master mix to each 8-µL hybridization reaction.

7. Flick the tube or slowly pipet up and down a few times to mix well, then spin
briefly.

8. Incubate the 20-µL ligation reaction in a thermal cycler at 16°C for 16 hours.
Note: If possible, set the temperature of the thermal cycler lid to match the block
temperature. Otherwise, incubate the reaction with the heated lid turned off, or
do not cover the reaction tubes with the heated lid.

Perform reverse Use components from the SOLiD™ Total RNA-Seq Kit:
transcription • Nuclease-free Water
• 10✕ RT Buffer
• dNTP Mix
• SOLiD™ RT Primer
• ArrayScript™ Reverse Transcriptase

1. Prepare RT master mix (without the ArrayScript™ Reverse Transcriptase):

Component Volume for one reaction†

Nuclease-free Water 11 µL
10X RT Buffer 4 µL
dNTP Mix 2 µL
SOLiD™ RT Primer 2 µL
Total volume per reaction 19 µL
† Include 5–10% excess volume in the master mix to compensate for pipetting error.

2. Incubate the RT master mix with the ligated RNA sample:

a. Add 19 µL of RT master mix to each 20-µL ligation reaction.
b. Pipet up and down a few times to mix, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 70°C for 5 minutes, then
snap-cool on ice.

40 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Construct the amplified low-input whole transcriptome library 3

3. Perform the reverse transcription reaction:

a. Add 1 µL ArrayScript™ Reverse Transcriptase to each ligated RNA sample.
b. Gently vortex to mix thoroughly, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 42°C for 30 minutes.

Note: The cDNA can be stored at –20°C for a few weeks, stored at –80°C for long-term
storage, or used immediately.

Purify and size- Using Agencourt® AMPure® XP, perform two rounds of bead capture, wash, and
select the cDNA: elution to ensure complete capture and size-selection of the desired cDNA.
round 1 The first round of bead capture binds cDNA products greater than 100 bp with a 1.8✕
addition of bead volume to the sample mixture. The subsequent washes remove
fragments that are less than 100 bp. This process increases the percentage of library
inserts that are in the desired size range.
Required materials:
• Agencourt® AMPure® XP Reagent (place at room temperature 30 minutes before
using)
• U-bottom microplate
• Magnetic Stand-96
• 70% ethanol, freshly prepared from 100% ethanol, ACS reagent grade or
equivalent
• Nuclease-free Water

1. Bind the cDNA to the beads:

a. Gently shake the Agencourt® AMPure® XP Reagent bottle to resuspend any
magnetic particles that may have settled.
b. Add 72 µL Agencourt® AMPure® XP Reagent to each 40 µL sample.
c. Transfer the 112-µL bead and sample mixture to a U-bottom microplate.
d. Mix thoroughly by pipette mixing 10 times. The color of the mixture should
appear homogenous after mixing.
e. Incubate samples for 5 minutes at room temperature.

2. Remove the buffer from the beads:

a. Place the U-bottom microplate onto a Magnetic Stand-96 for 2-5 minutes to
separate beads from the solution. Wait for the solution to clear before
proceeding to the next step.
b. While the plate is in the magnetic stand, aspirate the supernatant from the
plate and discard.

IMPORTANT! Do not disturb the magnetic beads. If beads are drawn out
during aspiration, leave a few microliters of supernatant behind.

SOLiD® Total RNA-Seq Kit 41

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Construct the amplified low-input whole transcriptome library

3. Wash the beads with 70% ethanol:

a. While the plate is on the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the U-bottom microplate and incubate for 30 seconds at room
temperature.
b. Aspirate the ethanol and discard.
c. While the plate is on the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the plate and incubate for 30 seconds at room temperature.
d. Aspirate the ethanol and discard.
e. Air dry the beads at room temperature for 5 minutes to remove all traces of
ethanol. Do not overdry beads (overdried beads appear cracked).
Note: Overdrying beads significantly decreases elution efficiency, however,
leftover ethanol may inhibit PCR.
f. Remove the plate from the Magnetic Stand-96.

4. Elute the cDNA from the beads:

a. Add 40 µL of Nuclease-free water to each well of the plate and pipette mix 10
times.
Note: Elution occurs quickly. It is not necessary for the beads to go back into
solution for it to occur. Some beads may appear clumpy.
b. Place the U-bottom plate on the Magnetic Stand-96 for 1 minute to separate
beads from the solution.
c. Transfer 40 µL eluant to an empty well of a U-bottom plate for the second
round of purification and size-selection. Then remove plate from Magnetic
Stand-96.
Note: You may use the same U-bottom plate for both rounds of purification and size-
selection. To avoid contamination, leave an empty row of wells between the two
rounds.

Purify and size- The second round of bead capture using Agencourt® AMPure® XP binds cDNA
select the cDNA: products greater than 150 bp with a 1.6✕ addition of bead volume to the sample
mixture. Two rounds of size selection are required to increase the percentage of library
round 2
inserts that are in the desired size range.

1. Bind the cDNA from the first bead capture to the beads:
a. Gently shake the Agencourt® AMPure® XP Reagent bottle to resuspend any
magnetic particles that may have settled.
b. Add 64 µL Agencourt® AMPure® XP Reagent to each 40 µL of sample from
the first bead capture.
c. Mix thoroughly by pipette mixing 10 times. The color of the mixture should
appear homogenous after mixing.
d. Incubate samples for 5 minutes at room temperature to bind cDNA products
to the magnetic beads.

42 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Construct the amplified low-input whole transcriptome library 3

2. Remove the buffer from the beads:

a. Place the U-bottom microplate onto a Magnetic Stand-96 for 2-5 minutes to
separate beads from the solution. Wait for the solution to clear before
proceeding to the next step.
b. While the plate is in the magnetic stand, aspirate the supernatant from the
plate and discard.

IMPORTANT! This step must be performed while the reaction plate is on the
magnetic stand. Do not disturb the beads. If necessary, leave a few
microliters of supernatant rather than disturb the beads.

3. Wash the beads with 70% ethanol:

a. While the plate is on the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the U-bottom microplate and incubate for 30 seconds at room
temperature.
b. Aspirate the ethanol and discard.
c. While the plate is on the magnetic stand, dispense 200 µL of 70% ethanol to
each well of the plate and incubate for 30 seconds at room temperature.
d. Aspirate the ethanol and discard.
e. Air dry the beads at room temperature for 5 minutes to remove all traces of
ethanol. Do not overdry beads (overdried beads appear cracked).
Note: Overdrying beads significantly decreases elution efficiency, however,
leftover ethanol may inhibit PCR.
f. Remove the plate from the Magnetic Stand-96.

4. Elute the cDNA:

a. While the plate is off the magnetic stand, add 40 µL of Nuclease-free water to
each well of the plate and pipette mix 10 times.
Note: Elution occurs quickly. It is not necessary for the beads to go back into
solution for elution to occur. Some beads may appear clumpy.
b. Place the U-bottom plate on the Magnetic Stand-96 for 1 minute to separate
beads from the solution. Wait for the solution to clear before proceeding to
the next step.
c. Transfer the eluant (40 µL) to a new 0.2-mL tube.

Amplify the cDNA Use components from the SOLiD™ Total RNA-Seq Kit:
• Nuclease-free Water
• 10✕ PCR Buffer
• dNTP Mix
• SOLiD™ 5′ PCR Primer
• AmpliTaq® DNA Polymerase
• SOLiD™ 3′ PCR Primer
(Optional) To prepare cDNA libraries for multiplex SOLiD System sequencing,
substitute with the barcoded SOLiD™ 3′ Primers from one of the SOLiD™ RNA
Barcoding Kits.

SOLiD® Total RNA-Seq Kit 43

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Construct the amplified low-input whole transcriptome library

Note: Plan your experiments to include multiples of four different barcoded

libraries in every multiplex sequencing pool, to preserve color balance on the
SOLiD System sequencing run. Use the SOLiD 3′ Primers from the SOLiD RNA
Barcoding Kit in the appropriate color-balanced groups. For more information,
refer to the product insert for your SOLiD RNA Barcoding Kit and the Applied
Biosystems SOLiD™ 4 System SETS Software User Guide (PN 4448411).

1. Transfer 20 µL of the eluted cDNA from the second round of purification and
size-selection to a new tube.

2. For each low input cDNA sample, prepare 29 µL PCR master mix:

Volume
for one
Component
reaction
(µL)†

Nuclease-free water 18.4 µL

10✕ PCR Buffer 5 µL
dNTP Mix 4 µL
SOLiD™ 5’ PCR Primer 1 µL
AmpliTaq® DNA Polymerase 0.6 µL
Total Volume 29 µL
† Include 5–10% excess volume in the master mix to compensate for pipetting
error.

3. Transfer 29 µL PCR master mix into each 20 µL cDNA sample.

4. Add 1 µL SOLiD 3’ PCR primer to each tube.
Note: To prepare cDNA libraries for multiplex SOLiD™ System sequencing,
substitute with the barcoded SOLiD™ 3’ PCR Primers from your SOLiD™ RNA
Barcoding Kit.

5. Run the PCR reactions in a thermal cycler:

Stage Temp Time

Hold 95°C 5 min
95°C 30 sec
Cycle (18 cycles) 62°C 30 sec
72°C 30 sec
Hold 72°C 7 min

44 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Construct the amplified low-input whole transcriptome library 3

Purify the Use the Invitrogen PureLink® PCR Micro Kit:

amplified DNA • PureLink® PCR Micro Kit Column
• Collection Tube
• Binding Buffer (B2)
• Wash Buffer (W1)
• PureLink® Elution Tube

IMPORTANT! Do not use other PCR purification kits. Other purification kits are not as
effective in the removal of unincorporated primers. Unincorporated primers can affect
the final quantitation and emulsion PCR.

1. Before using the PureLink® PCR Micro Kit Column, place it in a clean Collection
Tube, then spin the column at 10,000 × g for 1 minute.

2. Prepare the sample:

a. Transfer the 50-µL PCR reaction into a new 1.5-mL tube.

IMPORTANT! If you used two different barcoded SOLiD 3′ Primers for your
sample, do not combine the PCRs at this step.

b. Add 200 µL of Binding Buffer (B2) to the tube, then mix well.

3. Load the sample onto the PureLink® PCR Micro Kit Column:
a. Load 250 µL of the sample containing Binding Buffer (B2) onto the
PureLink® PCR Micro Kit Column.
b. Spin the column at 10,000 × g for 1 minute.
c. Discard the flowthrough.

4. Wash the DNA:

a. Return the column to the Collection Tube.
b. Add 600 µL of Wash Buffer (W1) to the column.
c. Spin the column at 10,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the column to the Collection Tube.
f. Spin the column at 14,000 × g for 1 minute.

5. Elute the DNA in a clean PureLink® Elution Tube:

a. Place the column in a clean PureLink® Elution Tube.
b. Add 10 µL of Elution Buffer to the center of the membrane.
c. Wait 1 minute, then spin the column at 14,000 × g for 1 minute.
d. Repeat step 5b through step 5c for a total elution volume of 20 µL.

SOLiD® Total RNA-Seq Kit 45

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Construct the amplified low-input whole transcriptome library

Assess the yield Assess the yield and size distribution of the low input amplified DNA using the
and size Agilent® DNA 1000 Kit (Agilent®). For instructions on how to perform a smear
analysis, see page 88.
distribution of the
amplified DNA 1. Measure the concentration of the purified DNA with a NanoDrop®
spectrophotometer, and if necessary, dilute the DNA to <50 ng/µL for accurate
quantitation with the DNA 1000 Kit.

2. Run 1 µL of the purified DNA on an Agilent® 2100 Bioanalyzer™ Instrument with

the DNA 1000 Kit. Follow the manufacturer’s instructions for performing the
assay.

3. Using the 2100 expert software, perform a smear analysis to quantify the
percentage of DNA that is 25–200 bp.
Note: In general, we see a positive correlation between RNA-Seq mapping
statistics and the percent of DNA library inserts that are above 200 bp in length.
We recommend minimizing the percent of inserts that fall in the 25-200 bp range.
Please use your judgement when deciding the best way to proceed with libraries
from precious samples that fall close to these recommended quality values of
more than 50% DNA in 25-200 bp range.

Percent of DNA in
Next steps
the 25–200 bp range

Less than 50% Proceed with SOLiD™ System templated bead

preparation (see page 47).
50-60% You may perform another round of purification and size-
selection on the amplified DNA using Agencourt®
AMPure® XP:
1. Bring the sample volume to 40 µL with Nuclease-free
Water.
2. Follow the instructions in “Purify and size-select the
cDNA: round 2” on page 42, using AMPureXP bead
purification to decrease the % of amplified DNA in the
25-200bp range. However, in order to maintain a high
concentration, elute with 20 µL of water, instead of
with the 40 µL used in step 4 of the procedure.
OR
Alternatively, you may proceed with SOLiD System
templated bead preparation and expect to see slightly
higher % of filtered reads in your sequencing data
when compared to libraries with less than 50% in the
25-200bp range.
More than 60% It is recommended that you perform another round of
purification and size-selection using Agencourt®
AMPure® XP. See step 1 and step 2 above.

Note: For instructions on how to perform the smear analysis, see “Perform a
smear analysis” on page 88. Refer to the Agilent® 2100 Bioanalyzer™ 2100 Expert
User’s Guide by Agilent®.

46 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Construct the amplified low-input whole transcriptome library 3

4. Determine the median peak size (bp) and molar concentration (nM) of the cDNA
library using the Agilent® software. For more information, see “Determine the
median size” on page 89.
Note: It is recommended that the amplified DNA be <50 ng/µL for accurate
quantitation with the DNA 1000 Kit. (DNA 1000 Kit has a quantitative range of
0.1-50 ng/µL)

Alternatively, obtain the mass concentration by another method, and convert the
mass concentration to molar concentration.

Note: If the sample concentration is too low, reassess the yield and size distribution
using the Agilent® High Sensitivity DNA Kit. (HS DNA Assay has a quantitative range
of 5-500 pg/µL)

Proceed with When less than 50% (or in some cases, less than 60%)of the amplified DNA is in the 25–
SOLiD™ System 200 bp range, you can proceed with the SOLiD™ System templated bead preparation
stage, in which each library template is clonally amplified on SOLiD™ P1 DNA Beads
templated bead
by emulsion PCR. Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead
preparation Preparation Guide (PN 4448378) or SOLiD™ EZ Bead Emulsifier Getting Started Guide
(Part no. 4441486
Barcoded libraries are pooled prior to templated bead preparation. For more
information refer to the product insert for your SOLiD™ RNA Barcoding Kit.
Note: When optimizing SOLiD Total RNA-Seq Kit library concentrations (singleplex
or multiplex sequencing pools) by workflow analysis (WFA), library concentrations of
0.4 pM and 0.8 pM for ePCR are recommended.

Typical size profiles Typical size distributions (Agilent® 2100 Bioanalyzer™ Instrument profiles) of
of amplified amplified libraries prepared from HeLa poly(A) RNA (Figure 9) using the SOLiD™
Total RNA-Seq Kit are shown.
libraries
Figure 9 Size distribution of amplified library prepared from the 5 ng HeLa poly(A) RNA

SOLiD® Total RNA-Seq Kit 47

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Troubleshooting

Troubleshooting
See “Troubleshooting” on page 31.

Using a positive A general troubleshooting strategy is to perform the SOLiD™ Total RNA-Seq Kit
control procedure using the WT Control RNA (HeLa total RNA) provided with the kit.
Use 5-25 ng WT Control RNA for the fragmentation procedure starting on page 35.

Typical results Mapping statistics comparing the Low Input Workflow to the Standard input (Traditional
In-Gel or AMPureXP bead size selection).

Total %Uniquely
RNA Sample cDNA Size %rRNA Number of Uniquely Mapped
Number of Mapped
Input Selection method Reads Reads
Reads Reads

5ng HeLa 2 Rounds of 30,202,243 3.89% 31.66% 9,561,753.75

PolyA_replicate 1 AMPureXP Bead
Purification
5ng HeLa 25,405,894 4.35% 38.58% 9,801,834.61
PolyA_replicate 2
500ng HeLa Traditional In-Gel 27,086,281 3.73% 56.62% 15,336,725.72
PolyA_replicate 1 (6% TBE-Urea Gel)
500ng HeLa 28,385,910 3.74% 56.03% 15,904,411.97
PolyA_replicate 2
500ng HeLa 2 Rounds of 29,963,374 3.26% 54.58% 16,353,049.01
PolyA_replicate 1 AMPureXP Bead
Purification
500ng HeLa 26,244,873 3.38% 57.51% 15,092,330.29
PolyA_replicate 2

Uniquely mapped reads for low input with 5ng Poly(A) RNA are ~20% lower
compared to the standard input at 500ng Poly(A). This is an attribute that is expected
with the decrease in expression complexity of low input amounts of Poly(A) RNA
when compared to the standard input. However, this decrease in % of uniquely
mapped reads does not negatively impact % detection of RefSeq transcripts (graph
below). In addition, low input Spearman correlations (by RefSeq RPKM) to the
standard in-gel method are greater than 0.9625. (data not shown)

48 SOLiD® Total RNA-Seq Kit

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
Troubleshooting 3

% RefSeq Transcripts Detected

SOLiD® Total RNA-Seq Kit 49

 Chapter 3 Whole Transcriptome Library Preparation: Low Input
3 Troubleshooting

50 SOLiD® Total RNA-Seq Kit

 4 Small RNA Library Preparation

Prepare the starting material

Assess the amount and quality of small RNA in your total RNA samples
(page 52)

Enrich the sample for small RNA (page 53)

Assess the quality and quantity of the small RNA-enriched sample (page 54)

Determine the input amount (page 54)

Construct the amplified small RNA library

Hybridize and ligate the RNA (page 55)

+ NNNNN B NNNNN NNNNN B NNNNN

Perform reverse transcription (page 56)

Purify the cDNA (page 56)

Size select the cDNA (page 57) cDNA

5′ PCR Primer

Amplify the cDNA (page 60) (Barcode)

optional

3′ PCR Primer

P1 sequence internal adaptor (IA) P2 sequence

Purify the amplified DNA (page 61) RNA sequence barcode (BC)

Assess the yield and size distribution of the amplified DNA (page 62)

Proceed with SOLiD™ System templated bead preparation

Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead Preparation P1
Guide (PN 4448378) or to IA BC
P2
SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no. 4441486)

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 Chapter 4 Small RNA Library Preparation
4 Prepare the starting material

Prepare the starting material

Preparing the starting material involves the following procedures:

1. Assess the amount and quality of small RNA in your total RNA samples (below)
2. Enrich the sample for small RNA (page 53)
3. Assess the quality and quantity of the small RNA-enriched sample (page 54)
4. Determine the input amount (page 54)

Guidelines for For this protocol, the total RNA must contain the small RNA fraction (microRNA or
obtaining small miRNA, 10–40 nt). For optimal results, use RNA that has been enriched for miRNA.
RNA We recommend using the following products:
• Recommended RNA source: Use Ambion FirstChoice® Total RNA, which is
certified to contain miRNA and other small RNAs.
• Recommended RNA isolation kits: Use the Ambion mirVanaTM miRNA Isolation
Kit or the Ambion mirVana PARISTM Kit to isolate small RNA or total RNA that
includes the small RNA fraction. Follow the procedure on page 91 to enrich small
RNA from total RNA using the Invitrogen PureLink® miRNA Isolation Kit.
• ERCC spike in RNAs should not be used when constructing small RNA libraries.
The ERCC RNAs will not be properly represented in the small RNA libraries
because they are outside of the size range of RNAs selected for these libraries.

Assess the amount Before you prepare the library, determine the quality of the total RNA sample. Use the
and quality of small NanoDrop® Spectrophotometer and the Agilent® 2100 Bioanalyzer™ Instrument with
the RNA 6000 Nano Kit and the Small RNA Kit.
RNA in your total
RNA samples 1. Quantitate the amount of RNA in the sample using the NanoDrop®
Spectrophotometer.
Note: If you used the PureLink® miRNA Isolation Kit to isolate small RNA from
samples, you can skip to step 1 on page 54.

2. Determine the quality and percentage of small RNA in your sample:

a. Dilute the sample to ~50 to 100 ng/µL.
b. Run 1 µL of diluted sample using the RNA 6000 Nano Kit with the Agilent®
2100 Bioanalyzer™ Instrument to determine the concentration of total RNA.
Follow the manufacturer’s instructions for performing the assay.
c. Using the 2100 expert software, determine the mass of total RNA in the
sample, and save for step 3c.
Note: Best mapping results are seen when libraries are prepared from input
RNA with RIN values ≥6. Use highest quality RNA available for library
preparation. Libraries can be prepared from RNA with lower RIN values for
precious samples. RNA degradation products may affect the quantitation of
small RNA in the sample and compete with small RNA ligation affecting the
number of sequencing reads uniquely mapping to miRBase (for instance).

52 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Prepare the starting material 4

3. Determine the percentage of small RNA in your sample:

a. Run 1 µL of diluted RNA on the Agilent® 2100 Bioanalyzer™ Instrument
with the Small RNA Chip. Follow the manufacturer’s instructions for
performing the assay.
b. Using the 2100 expert software, determine the mass of small RNA (miRNA,
10–40 nts) from the Small RNA Chip.
c. Calculate the miRNA content in your RNA sample using the following
formula:

% miRNA =
( mass of miRNA (10–40 nts) from the Small RNA Chip

mass of total RNA from the RNA 6000 Nano Chip ) x 100

4. Determine whether small RNA enrichment is needed:

How much miRNA

Recommendations for small RNA enrichment and next
(10–40 nt) is in your
steps
RNA sample?

≥0.5% miRNA You can use the total RNA in the ligation reaction, and small
RNA enrichment is not needed. However, for optimal results,
We recommend enrichment of all total RNA samples.
Proceed with “Enrich the sample for small RNA” on page 53
or skip to “Determine the input amount” on page 54.
<0.5% miRNA Small RNA enrichment is strongly recommended. We
recommend using the Invitrogen PureLink® miRNA
Isolation Kit.
Proceed with “Enrich the sample for small RNA” on page 53.

Guidelines for The amount of small RNA in samples varies widely according to the tissue source and
enriching for small the RNA isolation method. A survey by Agilent® provides a guide for the relative
proportion of miRNA of 40 different tissues (Tissot, 2008). If the tissues or cell lines
RNA
you are using contain a small fraction of small RNA, we recommend that you enrich
the RNA samples for small RNA.

Enrich the sample If needed, enrich the sample for small RNA using the method below:
for small RNA • If your RNA sample contains <0.5% miRNA – Use the Invitrogen PureLink®
miRNA Isolation Kit. Follow the instructions in “Small RNA enrichment” on
page 91.

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 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

Assess the quality Assess the quality and quantity of samples that are enriched for small RNA. Use the
and quantity of the Agilent® 2100 Bioanalyzer™ Instrument with the Small RNA Chip Kit (Agilent®).
small RNA- 1. Run 1 µL of enriched small RNA sample on the Agilent® 2100 Bioanalyzer™
enriched sample Instrument with the Small RNA Chip. Follow the manufacturer’s instructions for
performing the assay.

2. Compare the bioanalyzer traces to those of the sample before enrichment (step 2
in “Assess the amount and quality of small RNA in your total RNA samples” on
page 52), and determine whether the RNA is degraded.

3. Using the 2100 expert software, determine the quality and quantity of recovered
small RNA.

% miRNA =
( mass of miRNA (10–40 nts) from the Small RNA Chip

mass of enriched small RNA from the Small RNA Chip ) x 100

For enriched small RNA samples, peaks should be from 10 to 200 nt.

Determine the Using the following formula, determine the amount of total RNA to use according to
input amount the type of RNA you ran and the percentage of miRNA in the RNA sample.

the amount of miRNA

the amount of total RNA input =
%miRNA

Total RNA
Input sample type Amount of miRNA (10 to 40 nt)
input†

Total RNA 5 to 100 ng ≤1 µg

Enriched small RNA 1 to 100 ng ≤1 µg
† The yield drops if you use more than 1 µg of RNA for ligation.

Construct the amplified small RNA library

Constructing the amplified small RNA library involves the following procedures:

1. Hybridize and ligate the RNA (page 55)

2. Perform reverse transcription (page 56)
3. Purify the cDNA (page 56)
4. Size select the cDNA (page 57)
5. Amplify the cDNA (page 60)
6. Purify the amplified DNA (page 61)
7. Assess the yield and size distribution of the amplified DNA (page 62)
8. Proceed with SOLiD™ System templated bead preparation (page 63)

54 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Construct the amplified small RNA library 4

Hybridize and Use components from the SOLiD™ Total RNA-Seq Kit:
ligate the RNA • Hybridization Solution
• Nuclease-free Water
• SOLiD™ Adaptor Mix
• 2✕ Ligation Buffer
• Ligation Enzyme Mix

1. On ice, prepare the hybridization mix in 0.5-mL PCR tubes:

Component Volume (µL)

Small RNA sample: 3
• 5 to 100 ng of miRNA in ≤1 µg of total RNA
• 1 to 100 ng of miRNA in ≤1 µg of enriched small
RNA
Hybridization Solution 3
SOLiD™ Adaptor Mix 2
Total volume per reaction 8

2. Slowly pipet up and down a few times to mix well, then spin briefly.
3. Run the hybridization reaction in a thermal cycler:

Temperature Time
65°C 10 min
16°C 5 min

4. Add the RNA ligation reagents to the 8-µL hybridization reactions:

Component (add in order shown) Volume (µL)

2✕ Ligation Buffer 10
Ligation Enzyme Mix 2

IMPORTANT! You may observe a white precipitate in the 2✕ Ligation Buffer. If so,
warm the tube at 37°C for 2–5 minutes or until the precipitate is dissolved.
2✕ Ligation Buffer is very viscous; pipet slowly to dispense it accurately.

5. Flick the tube or slowly pipet up and down a few times to mix well, then spin
briefly.

6. Incubate the 20-µL ligation reaction in a thermal cycler at 16°C for 16 hours.

IMPORTANT! Turn off the heated lid or leave the thermal cycler open during the
incubation.

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 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

Perform reverse Use components from the SOLiD™ Total RNA-Seq Kit:
transcription • Nuclease-free Water
• 10✕ RT Buffer
• 2.5 mM dNTP Mix
• SOLiD™ RT Primer
• ArrayScript™ Reverse Transcriptase

1. On ice, prepare RT master mix (without the ArrayScript™ Reverse Transcriptase):

Volume for
Component each reaction
(µL)†
Nuclease-free Water 11
10X RT Buffer 4
dNTP Mix 2
SOLiD™ RT Primer 2
Total volume per reaction 19
† Include 5–10% excess volume in the master mix to compensate for pipetting error.

2. Incubate the RT master mix with the ligated RNA sample:

a. Add 19 µL of RT master mix to each 20-µL ligation reaction.
b. Pipet up and down a few times to mix, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 70°C for 5 minutes, then
snap-cool on ice.

3. Perform the reverse transcription reaction:

a. Add 1 µL ArrayScript™ Reverse Transcriptase to each ligated RNA sample.
b. Gently vortex to mix thoroughly, then spin briefly.
c. Incubate in a thermal cycler with a heated lid at 42°C for 30 minutes.

Note: The cDNA can be stored at –20°C for a few weeks, stored at –80°C for long-term
storage, or used immediately.

Purify the cDNA Use the MinElute® PCR Purification Kit (Qiagen).
Note: The kit may be supplied with Buffer PB (without pH Indicator) or Buffer PBI
(with pH Indicator). Either buffer can be used as is; it is not necessary to add pH
Indicator to Buffer PB before use.

1. Add Nuclease-free Water and Buffer PB or Buffer PBI to the cDNA:

a. Transfer all of the cDNA (40 µL) to a clean 1.5-mL microcentrifuge tube.
b. Add 60 µL of Nuclease-free Water.
c. Add 500 µL of Buffer PB or Buffer PBI, then mix well.

56 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Construct the amplified small RNA library 4

2. Load the cDNA onto the MinElute column:

a. Load 600 µL of the sample containing Buffer PB or Buffer PBI onto the
MinElute column.
b. Spin the column at 13,000 × g for 1 minute.
c. Discard the flowthrough.

3. Wash the cDNA:

a. Return the MinElute column to the microcentrifuge tube.
b. Add 750 µL of Buffer PE to the MinElute column.
c. Spin the column at 13,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the MinElute column to the microcentrifuge tube.
f. Spin the column at 13,000 × g for 1 minute.

4. Elute the cDNA in a clean microcentrifuge tube:

a. Place the MinElute column in a clean microcentrifuge tube.
b. Add 10 µL of Buffer EB to the center of the MinElute column.
c. Wait 1 minute, then spin the column at 13,000 × g for 1 minute.

Size select the Perform gel purification to separate library products from unused adaptors and
cDNA primers. The gel purification method yields precise size-selection and the highest
quality mapping.
Use Invitrogen Novex® pre-cast gel products, a 10 bp DNA Ladder, and SYBR® Gold
nucleic acid gel stain:
• Invitrogen Novex® 10% TBE-Urea Gel 1.0 mM, 10 Well
• Invitrogen Novex® TBE Running Buffer (5✕)
• Invitrogen Novex® TBE-Urea Sample Buffer (2✕)
• Invitrogen XCell SureLock™ Mini-Cell
• Invitrogen 10 bp DNA Ladder
• Invitrogen SYBR® Gold nucleic acid gel stain
For more instructions on running Novex gels, refer to the Novex® Pre-Cast Gel
Electrophoresis Guide. For more instructions on staining the gel, refer to the SYBR® Gold
Nucleic Acid Gel Stain manual.

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 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

1. Prepare the gel as described in the Novex® Pre-Cast Gel Electrophoresis Guide:
a. Prepare 1000 mL of 1✕ TBE Running Buffer:

Component Volume (mL)

Novex® TBE Running Buffer (5✕) 200
Deionized water 800
Total volume 1000

b. Place the Novex® 10% TBE-Urea Gel in the XCell SureLock™ Mini-Cell.
c. Add 1✕ TBE Running Buffer to the Upper Buffer Chamber and the Lower
Buffer Chamber.

2. Dilute the 10-bp DNA Ladder:

Component Volume (µL) Concentration

10-bp DNA Ladder 1 1 µg/µL
RNase-free water 24 –
Total volume 25 40 ng/µL

3. Prepare the cDNA and the DNA ladder:

a. Mix 5 µL of the cDNA with 5 µL of 2✕ Novex TBE-Urea Sample Buffer.
b. Mix 5 µL of the 40 ng/µL 10 bp DNA Ladder with 5 µL of 2✕ Novex TBE-
Urea Sample buffer.
c. Heat the cDNA and the DNA Ladder at 95°C for 3 minutes.
d. Snap-cool the tubes on ice. Leave the tubes on ice for less than 30 minutes.
Note: Do not leave denatured samples on ice for longer than 30 minutes. If
the denatured samples are left on ice for longer than 30 minutes, repeat
step 3c before loading the samples.

4. Before you load the samples, flush the wells of the gel several times with 1X TBE
Running Buffer to remove urea from the wells.
Note: Flushing the wells is important to obtain sharp bands.

5. Load the cDNA samples and the DNA Ladder.

Note: Follow these guidelines when loading the gel:
• Do not use the lanes next to the edges of the gel (lanes #1 and #10).
• Load the DNA Ladder on both sides of each cDNA sample to help you make
accurate cuts.
• While loading, place the pipette tip as close to the bottom of the well as
possible, and load the sample slowly. It is important to keep the sample
compact in the gel.

58 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Construct the amplified small RNA library 4

6. Run the gel at 180 V until the second dye front just passes the middle of the gel
(~45 minutes).

Run the gel until the second dye front just

passes the middle of the gel

IMPORTANT! Shortening or lengthening the running time results in inferior

resolution.

7. Add 5 µL of the SYBR Gold nucleic acid gel stain to 50 mL of 1✕ TBE Running
Buffer, then stain the gel for 5–10 minutes.

8. Illuminate the stained gel, then excise the gel containing 60 to 80 nt of cDNA:
Note: Be careful not to include extra gel that does not contain any cDNA.
a. Using a clean razor blade, make horizontal cuts directly on the 60-nt and
80-nt bands to excise the gel between 60 and 80 nt of cDNA.

Make the horizontal cuts first to obtain the

desired insert length

Note: If you are using a UV transilluminator to visualize the reaction

products, work quickly to limit their exposure to UV radiation.
Note: To obtain the desired insert length, you can adjust the cuts. However,
too much gel will severely inhibit the PCR reactions. See page 60 for the
expected lengths of the insert and PCR product according to the length of the
cDNA that is excised from the gel.

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 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

b. Reduce the width of the gel piece by making vertical cuts on both edges of
the smear.

Make the vertical cuts on both edges of the

smear

9. Transfer the gel piece to a clean working area, maintaining the orientation of the
gel, then cut the gel vertically into 4 pieces using a clean razor blade. Each gel slice
should be about 1 mm × 6 mm.

Cut the gel vertically into 4 pieces

10. Place the two gel slices from the middle of the lane individually into clean 0.2-mL
PCR tubes, and place the outside gel slices into a clean 1.5-mL microcentrifuge
tube for storage.
To generate sufficient cDNA for accurate quantitation, you need to run 2
amplification reactions using 2 gel pieces, 1 gel piece in each reaction. You may
store the other 2 pieces for 2 weeks at –20°C.
Note: To maximize the yield for SOLiD System sequencing, use the 2 gel pieces
from the middle of the lane.

Expected lengths
Excised cDNA length (nt) Insert length (bp) PCR product length (bp)
of the insert and
PCR product 42 ~0 ~93
according to 60 ~18 ~110
excised cDNA 80 ~38 ~130
length

Amplify the cDNA Use components from the SOLiD™ Total RNA-Seq Kit:
• Nuclease-free Water
• 10✕ PCR Buffer
• 2.5 mM dNTP Mix
• SOLiD™ 5′ PCR Primer
• AmpliTaq® DNA Polymerase
• SOLiD™ 3′ PCR Primer
(Optional) To prepare cDNA libraries for multiplex SOLiD System sequencing,
substitute with the barcoded SOLiD™ 3′ Primers from one of the SOLiD™ RNA
Barcoding Kits.
Note: Plan your experiments to include multiples of four different barcoded
libraries in every multiplex sequencing pool, to preserve color balance on the
SOLiD System sequencing run. Use the SOLiD 3′ Primers from the SOLiD RNA
Barcoding Kit in the appropriate color-balanced groups. For more information,
refer to the product insert for your SOLiD RNA Barcoding Kit and the Applied
Biosystems SOLiD™ 4 System SETS Software User Guide (PN 4448411).

60 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Construct the amplified small RNA library 4

1. For each cDNA sample, prepare duplicate in-gel amplification reactions to

generate sufficient cDNA for emulsion PCR:
a. Ensure that each gel slice from step 10 on page 60 is placed in a 0.2-mL PCR
tube. If necessary, transfer the gel slice to the PCR tube using a clean pipette
tip.
b. For each cDNA sample, prepare 98 µL PCR mix for each gel slice:

Volume (µL)
Component
One 100-µL Two 100-µL
reaction reactions†

Nuclease-free Water 76.8 169.0

10✕ PCR Buffer 10.0 22.0
2.5 mM dNTP Mix 8.0 17.6
SOLiD™ 5′ PCR Primer 2.0 4.4
AmpliTaq® DNA Polymerase 1.2 2.6
Total volume 98.0 215.6
† Includes 10% excess volume to compensate for pipetting error.

c. Transfer 98 µL of PCR master mix into each 0.2-mL PCR tube.

d. Add 2 µL SOLiD 3′ PCR Primer to each tube.
Note: To prepare cDNA libraries for multiplex SOLiD System sequencing,
substitute with the barcoded SOLiD™ 3′ PCR Primers from your SOLiD™
RNA Barcoding Kit.

2. Run the PCR reactions in a thermal cycler:

Stage Temp Time

Hold 95°C 5 min
95°C 30 sec
Cycle (15 cycles) 62°C 30 sec
72°C 30 sec
Hold 72°C 7 min

Note: If you started with total RNA and input 1–25 ng, run 18 cycles. If you
started with enriched/purified small RNA and input 1–10 ng, run 18 cycles.

Purify the Use the Invitrogen PureLink® PCR Micro Kit:

amplified DNA • PureLink® Micro Kit Column
• Collection Tube
• Binding Buffer (B2)
• Wash Buffer (W1)
• PureLink® Elution Tube

SOLiD® Total RNA-Seq Kit 61

 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

IMPORTANT! Do not use other PCR purification kits. Other purification kits are not as
effective in the removal of unincorporated primers. Unincorporated primers can affect
the final quantitation and emulsion PCR.

1. Before using the PureLink® Micro Kit Column, place it in a clean Collection Tube,
then spin the column at 10,000 × g for 1 minute.

2. Prepare the sample:

a. Combine the two 100-µL PCR reactions in a new 1.5-mL tube.

IMPORTANT! If you used two different barcoded SOLiD 3′ Primers for your
sample, do not combine the PCRs at this step.

b. Add 800 µL of Binding Buffer (B2) to the tube, then mix well.

3. Load the sample onto the PureLink® Micro Kit Column:

a. Load 500 µL of the sample containing Binding Buffer (B2) onto the
PureLink® Micro Kit Column.
b. Spin the column at 10,000 × g for 1 minute.
c. Discard the flowthrough.
d. Load the remaining 500 µL of the sample containing Binding Buffer (B2)
onto the column.
e. Spin the column at 10,000 × g for 1 minute.
f. Discard the flowthrough.

4. Wash the DNA:

a. Return the column to the Collection Tube.
b. Add 600 µL of Wash Buffer (W1) to the column.
c. Spin the column at 10,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the column to the Collection Tube.
f. Spin the column at 14,000 × g for 1 minute.

5. Elute the DNA in a clean PureLink® Elution Tube:

a. Place the column in a clean PureLink® Elution Tube.
b. Add 12 µL of Elution Buffer to the center of the membrane.
c. Wait 1 minute, then spin the column at 14,000 × g for 1 minute.

Assess the yield Use the Agilent® 2100 Bioanalyzer™ Instrument with the DNA High Sensitivity Kit or
and size DNA 1000 Kit (Agilent®). For instructions on how to perform a smear analysis, see
page 88.
distribution of the
amplified DNA 1. Run 1 µL of the purified DNA on an Agilent® 2100 Bioanalyzer™ Instrument with
the DNA High Sensitivity Kit or DNA 1000 Kit. Follow the manufacturer’s
instructions for performing the assay.

62 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Construct the amplified small RNA library 4

2. Using the 2100 expert software, perform a smear analysis to determine whether
you can proceed with SOLiD™ System templated bead preparation:
a. Measure the area for the DNA that is 25–150 bp (the size range for ligation
products with no insert and ligation products with short inserts) and 120–
130 bp (the size range for the desired miRNA ligation products).
b. Calculate the ratio of 120–130-bp DNA: 25–150-bp DNA:
[Area (120–130 bp)]÷[Area (25–150 bp)]
Note: In general, we see a positive correlation between the percentage of
small RNA reads mapped to miRBase and the percentage of the DNA library
that is between 120-130 bp. We recommend maximizing the percentage of
inserts that fall into the 120-130 bp range. Please use your judgement when
deciding the best way to proceed with libraries from precious samples that
fall close to these recommended quality values.

Ratio of 120–130-bp
Next steps
DNA:25–150-bp DNA

Greater than 50% See “Proceed with SOLiD™ System templated bead
preparation” on page 63
Less than 50% Consider “Second-round size selection of amplified cDNA”
on page 85. Alternatively, proceed with templated bead prep
recommendations with potentially fewer sequencing reads
uniquely mapping to miRBase.

Note: Samples that are run on a bioanalyzer typically show 5 to 8 bp larger than their
actual size.

Proceed with You can proceed with the SOLiD™ System templated bead preparation stage, in which
SOLiD™ System each library template is clonally amplified on SOLiD™ P1 DNA Beads by emulsion
PCR. Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead Preparation Guide
templated bead
(Part. no. 4448378) or SOLiD™ EZ Bead Emulsifier Getting Started Guide
preparation (Part. no. 4441486).
Barcoded libraries are pooled prior to templated bead preparation. For more
information refer to the product insert for your SOLiD™ RNA Barcoding Kit.
Note: When optimizing SOLiD Total RNA-Seq Kit library concentrations (singleplex
or multiplex sequencing pools) by workflow analysis (WFA), library concentrations of
0.4 pM and 0.8 pM for ePCR are recommended.

SOLiD® Total RNA-Seq Kit 63

 Chapter 4 Small RNA Library Preparation
4 Construct the amplified small RNA library

Typical size profiles Figure 10 Size distribution of amplified library prepared from placenta total RNA
of amplified
libraries

Figure 11 Size distribution of amplified library prepared from HeLa total RNA and required a
second round of size selection

Figure 12 Sub-optimal size distribution of amplified library from partially degraded placenta
total RNA

64 SOLiD® Total RNA-Seq Kit

 Chapter 4 Small RNA Library Preparation
Troubleshooting 4

Troubleshooting

Observation Possible cause Solution

Agilent® software does not calculate The software detects multiple peaks in Refer to “Analyze multiple peaks as
one concentration and peak size the amplified cDNA profile one peak” on page 90.
Low yield in the desired size range and Your input amount is too low • Use enriched small RNA instead of
high background of small sizes total RNA for ligation.
(~100-bp and 115-bp by-products) • Use more RNA (<1 µg) for ligation.
• Perform another gel purification of
PCR products to select the desired
range (110–140 bps)
Low yield in the desired size range and tRNA is partially degraded Run the remaining 5 µL cDNA on a
high background of large sizes 10% TBU gel and select only 60–70-nt
(~135-bp by-products) (see Figure 12 sizes (see page 59).
on page 64)
Normal yield at desired size range but The gel selection step does not • Run the remaining cDNA on
background (100 bp, 115 bp) is too exclude small ligation products another gel; cut right on 60 nt so
high (see Figure 11 on page 64) that you do not include anything
smaller than the 18-nt insert.
• Perform another gel purification of
PCR products to select the desired
size range (110–140 bps).
Low yield and no PCR products The gel ran too long or too much gel Reduce the running time (step 6 on
was added to the PCR page 59) and add less gel to the PCR
(step 1 on page 61).
An enzymatic reaction or column 1. Dilute the cDNA 1:10, then use 1 µL
purification failed in a 50-µL PCR.
2. Check the yield before and after
purification using the PureLink®
PCR Micro Kit.
Low yield with miRNA input >25 ng. In rare cases, too much input inhibits Reduce the input amount to 5–10 ng of
the ligation reaction. miRNA (page 54}.
Normal or high yield but PCR products Too many PCR cycles resulted in Decrease the number of PCR cycles
larger than 150 bp overamplification (step 2 on page 61).
Normal yield and bad size distribution Too much sample was loaded on the Decrease the volume of sample
in the amplified library Novex® TBE-Urea Gel loaded to less than 10 µL (step 5 on
page 58).
The wells of the Novex TBE-Urea Gel Before you load the samples, flush the
contained urea wells of the gel several times with 1X
TBE Running Buffer to remove urea
from the wells and to obtain sharp
bands (step 4 on page 58).

SOLiD® Total RNA-Seq Kit 65

 Chapter 4 Small RNA Library Preparation
4 Troubleshooting

Using a positive A general troubleshooting strategy is to perform the SOLiD™ Total RNA-Seq Kit
control procedure using the small RNA control (Placenta total RNA) provided with the kit.
• Use 500 ng small RNA for hybridization and ligation starting on page 55.
• Use 5 µg small RNA control for small RNA enrichment starting on page 91.
Follow the instructions on page 56 to access the quality land quantity of small
RNA-enriched control sample and use 25 ng of miRNA for hybridization and
ligation.

66 SOLiD® Total RNA-Seq Kit

 A Ordering Information

How to order
For more information on the SOLiD™ Total RNA-Seq Kit and SOLiD™ RNA
Barcoding Kits, go to [Link].

Item Catalog/Part no.

SOLiD™ Total RNA-Seq Kit 4445374

SOLiD™ RNA Barcoding Kit, Modules 1-16 4427046
SOLiD™ RNA Barcoding Kit, Modules 17-32 4453189
SOLiD™ RNA Barcoding Kit, Modules 33-48 4453191
SOLiD™ RNA Barcoding Kit, Modules 49-64 4456501
SOLiD™ RNA Barcoding Kit, Modules 65-80 4456502
SOLiD™ RNA Barcoding Kit, Modules 81-96 4456503
SOLiD™ RNA Barcoding Kit, Modules 1-48 4461565
SOLiD™ RNA Barcoding Kit, Modules 49-96 4461566
SOLiD™ RNA Barcoding Kit, Modules 1-96 4461567

Optional materials and equipment not included

To order Ambion or Invitrogen products, go to [Link].

For whole
Item Catalog/Part no.
transcriptome
libraries (standard Ambion® 5 M Ammonium Acetate, 500 mL AM9071
input) Ambion® FirstChoice® Total RNA Go to
[Link]
Ambion® Gel Loading Solution (All-purpose, Native Agarose) AM8556
Ambion® Glycogen (5 mg/mL) (1 mL tube) AM9510
Ambion® MicroPoly(A)Purist™ Kit AM1919
Ambion® Spin Columns and Tubes AM10065
Ambion® TE, pH 8.0, 500 mL AM9849
Invitrogen Novex 6% TBE Gel, 1.0 mM, 10 well EC6265BOX
Invitrogen RiboMinus™ Eukaryote Kit for RNA-Seq A1083708

SOLiD® Total RNA-Seq Kit 67

 Appendix A Ordering Information
A Optional materials and equipment not included

Item Catalog/Part no.

Invitrogen RiboMinus™ Plant Kit for RNA-Seq A1083808

21-gauge needle Major laboratory
supplier (MLS)
Isopropanol Major laboratory
supplier (MLS)
ERCC RNA Spike-In Mix 4456740
ERCC ExFold RNA Spike-In Mixes 4456739

For whole
transcriptome Item Catalog/Part no.
libraries (low input)
Ambion® MicroPoly(A)Purist™ Kit AM1919
Invitrogen mRNA Catcher™PLUS Kit K1570-02
ERCC RNA Spike-In Mix 4456740
ERCC ExFold RNA Spike-In Mixes 4456739

For small RNA

Item Catalog/Part no.
libraries
Ambion® 5 M Ammonium Acetate, 500 mL AM9071
Ambion® ElectroZap™ Electrode Decontamination Solution AM9785
Ambion® FirstChoice® Total RNA Go to
[Link].
Ambion® Gel Loading Solution (All-purpose, Native Agarose) AM8556
Ambion® Glycogen (5 mg/mL) (1 mL tube) AM9510
Ambion® mirVana™ miRNA Isolation Kit, 40 purifications AM1560
Ambion® mirVana™ PARIS™, 40 purifications AM1556
Ambion® NucAway™ Spin Columns Kit, 30 each AM10070
Ambion® Spin Columns and Tubes AM10065
Ambion® TE, pH 8.0, 500 mL AM9849
Invitrogen Novex 6% TBE Gel, 1.0 mM, 10 well EC6265BOX
Invitrogen PureLink® miRNA Isolation Kit, 25 preps K1570-01
21-gauge needle Major laboratory
supplier (MLS)
Isopropanol Major laboratory
supplier (MLS)

68 SOLiD® Total RNA-Seq Kit

 Appendix A Ordering Information
Optional materials and equipment not included A

For whole
transcriptome Item Catalog no.
libraries (standard
Invitrogen™ XCell SureLock™ Mini-Cell EI0001
input): Gel-based
size selection Transilluminator Major laboratory
supplier (MLS)
Invitrogen™ 50 bp DNA Ladder 10416-014
Invitrogen™ Novex® 6% TBE-Urea Gels 1.0 mm, 10 well EC6865BOX
Invitrogen™ Novex® TBE Running Buffer (5✕), 1 L LC6675
Invitrogen™ Novex® TBE-Urea Sample Buffer (2✕), 10 mL LC6876
Invitrogen™ SYBR® Gold nucleic acid gel stain, 10,000✕ concentrate S-11494
in DMSO, 500 µL
MinElute® PCR Purification Kit (50) Qiagen 28004
(optional) ERCC RNA Spike-In Mix 4456740
(optional) ERCC ExFold RNA Spike-In Mixes 4456739

SOLiD® Total RNA-Seq Kit 69

 Appendix A Ordering Information
A Optional materials and equipment not included

70 SOLiD® Total RNA-Seq Kit

 B Supplemental Information

This appendix contains:

■ Amplified library construction concepts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
■ Sequences of the SOLiD™ primers included in the kit. . . . . . . . . . . . . . . . . . . . . . 72
■ About the RNA fragmentation methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
■ Gel size selection for Whole Transcriptome workflow standard input . . . . . . . . 75
■ Second-round size selection of amplified cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
■ Using 2100 expert software to assess whole transcriptome libraries . . . . . . . . . . 88
■ Small RNA enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
■ Using 2100 expert software to assess small RNA libraries. . . . . . . . . . . . . . . . . . . 93

Amplified library construction concepts

The procedures in this protocol are based on Applied Biosystems Ligase-Enhanced
Genome Detection (LEGenD™) technology (patent pending).

Hybridization and The RNA samples are hybridized and ligated with the SOLiD™ Adaptor Mix. The
ligation to the SOLiD™ Adaptor Mix is a set of oligonucleotides with a single-stranded degenerate
sequence at one end and a defined sequence required for SOLiD™ System sequencing
Adaptor Mix
at the other end. The SOLiD™ Adaptor Mix constrains the orientation of the RNA in
the ligation reaction such that hybridization with the SOLiD™ Adaptor Mix yields
template for SOLiD System sequencing from the 5′ end of the sense strand. The
downstream emulsion PCR primer alignment and the resulting products of templated
bead preparation for SOLiD System sequencing are illustrated in Figure 13.
Figure 13 Strand-specific RNA sequence information from SOLiD™ Total RNA-Seq Kit products

P1
Bead 5’ 3’
5’ 3’
RNA Sequence Internal Adapter barcode
3’ 5’
P2
5’ 3’
FORWARD

Reverse The RNA population with ligated adaptors is reverse transcribed to generate single-
transcription and stranded cDNA copies of the fragmented RNA molecules. cDNA with insert sizes
greater than 100 bp is selected by two rounds of Agencourt® AMPure® XP bead
size selection
capture, wash, and elution.

SOLiD® Total RNA-Seq Kit 71

 Appendix B Supplemental Information
B Sequences of the SOLiD™ primers included in the kit

cDNA library The size-selected cDNA is amplified using 15–18 cycles of PCR. This step appends
amplification required terminal sequences to each molecule and generates sufficient template for
SOLiD System sequencing. Limiting the cycle number minimizes the synthesis of
(single- or
spurious PCR products and better preserves the RNA profile of the sample.
multiplex) and final
cleanup • To prepare template for singleplex SOLiD System sequencing, use the PCR
primers included in the SOLiD Total RNA-Seq Kit.
• For multiplex SOLiD System sequencing, use the 3′ PCR primers supplied in a
SOLiD™ RNA Barcoding Kit.
The 3′ (reverse) PCR primers in the SOLiD™ RNA Barcoding Kits contain the P2
sequence required for SOLiD emulsion PCR, a unique barcode sequence, and an
internal adaptor sequence necessary for sequencing the barcode (Figure 13). Use a
different 3′ PCR primer in the amplification reaction for each cDNA sample to
generate a barcoded cDNA library that can be mixed with other barcoded
libraries for multiplex SOLiD System sequencing. Plan the in-gel PCRs so that
barcoded libraries are generated using multiples of the color-balanced groups of
four 3′ primers, to preserve color balance during multiplexed sequencing on the
SOLiD System. For more information, refer to the product insert for your
SOLiD™ RNA Barcoding Kit and Applied Biosystems SOLiD™ 4 System SETS
Software User Guide (PN 4448411) or SOLiD™ EZ Bead™ Amplifier Getting Started
Guide (Part no. 4443494).
The 5′ PCR primer is identical in each kit; its sequence corresponds to SOLiD emulsion
PCR primer 1 (P1 in Figure 13).
After the PCR, the amplified cDNA is cleaned up using the PureLink® PCR Micro Kit.
The yield and size distribution of each cDNA library is assessed; it is important to have
sufficient cDNA for accurate quantitation prior to SOLiD System templated bead
preparation, and to use only libraries with sufficiently long inserts for SOLiD System
sequencing.

Sequences of the SOLiD™ primers included in the kit

SOLiD™ 5′ PCR The 5' PCR primer is the SOLiD emulsion PCR primer P1.
primer 5′ - CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT -3′

SOLiD™ 3′ PCR 5′ - CTG CCC CGG GTT CCT CAT TCT CTG TGT AAG AGG CTG CTG TAC GGC
primer CAA GGC G -3′

72 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
About the RNA fragmentation methods B

About the RNA fragmentation methods

To generate whole transcriptome cDNA libraries with insert sizes suitable for SOLiD™
sequencing, you need to fragment the poly(A) RNA or rRNA-depleted total RNA to
100–200 nts before proceeding to ligation.
In this guide, we provide procedures for two methods of RNA fragmentation. Select
the fragmentation method from the two options presented by using the guidance
below:
• RNase III fragmentation: Provides convenience and robustness for
comprehensive transcriptome analysis using the SOLiD™ system. RNase III
digestion leaves 5′ phosphate and 3′ hydroxyl groups on the RNA fragments, and
the resulting RNA fragments can be directly used in the hybridization and
ligation reactions. The RNase III fragmentation method produces more consistent
sequencing results because this method is effective across a wide range of enzyme
activities, incubation times, and input amounts. We have observed RNaseIII
fragmentation results in lower amounts of rRNA fragments being sequenced than
when chemical fragmentation is used. This is assumed to be related to the role
RNaseIII has in processing rRNAs. This method is useful for performing
transcript-level quantification.
• Chemical fragmentation: The resulting RNA fragments need to be repaired using
T4 polynucleotide kinase before proceeding to ligation. Because RNA
fragmentation by chemical hydrolysis occurs rapidly, we recommend that you
determine the optimal conditions for your experimental system. As noted above,
we have observed a higher percentage of rRNA sequences are present in libraries
constructed from chemically fragmented RNA than with RNaseIII fragmented
RNA. Therefore, if minimizing rRNA sequences in the final library is very
important, RNaseIII should be considered for fragmentation. The chemical
fragmentation method produces a more uniform distribution of sequence tags
across the transcripts. This method is useful for quantifying exon levels,
identifying novel splicing, and discovering fusion genes.

SOLiD® Total RNA-Seq Kit 73

 Appendix B Supplemental Information
B Gel-based whole transcriptome library preparation: Standard Input

Gel-based whole transcriptome library preparation: Standard

Input

Fragment the whole transcriptome RNA (standard input)

Start with 100–500 ng poly(A) RNA or 200–500 ng rRNA-depleted total RNA

Fragment the RNA:

Fragment the RNA using RNase III (page 15) or
Fragment the RNA by chemical hydrolysis (page 16)
Fragmented RNA

Clean up the fragmented RNA (page 17)

Assess the yield and size distribution of the fragmented RNA (page 18)

Construct the amplified whole transcriptome library (standard input)

Hybridize and ligate the RNA (page 21) + NNNNN B NNNNN NNNNN B NNNNN

Perform reverse transcription (page 22)

Purify the cDNA (page 75)

Size select the cDNA (page 76) cDNA

5′ PCR Primer

Amplify the cDNA (page 80) (Barcode)

optional

3′ PCR Primer

P1 sequence internal adaptor (IA) P2 sequence

Purify the amplified DNA (page 81)
RNA sequence barcode (BC)

Assess the yield and size distribution of the amplified DNA (page 82)

Proceed with SOLiD™ System templated bead preparation

Refer to the Applied Biosystems SOLiD™ 4 System Templated Bead Preparation P1
Guide (PN 4448378) or to IA BC
P2
SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no. 4441486)

74 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Gel size selection for Whole Transcriptome workflow standard input B

Gel size selection for Whole Transcriptome workflow standard

input
Although bead size selection is convenient for removing inserts that are too short from
libraries, the size ranges of the final libraries are usually broad, ranging from 150 to 400
bps.
Gel size selection of cDNA generates libraries with more accurate, defined insert sizes.
The gel procedure below generates whole transcriptome libraries with insert sizes
from 100 to 200 bps. In addition, the insert size range can be adjusted by changing the
cutting range on the gel.
Note: See the workflow on page 74 for more information. Follow the procedures from
“Fragment the whole transcriptome RNA” on page 15 to “Hybridize and ligate the
RNA” on page 21 and “Perform reverse transcription” on page 22. Then proceed with
the following procedures for cDNA cleanup and gel size selection.

Purify the cDNA Note: The kit may be supplied with Buffer PB (without pH Indicator) or Buffer PBI
(with pH Indicator). Either buffer can be used as is; it is not necessary to add pH
Indicator to Buffer PB before use.

1. Add Nuclease-free Water and Buffer PB or Buffer PBI to the cDNA:

a. Transfer all of the cDNA (40 µL) to a clean 1.5-mL microcentrifuge tube.
b. Add 60 µL of Nuclease-free Water.
c. Add 500 µL of Buffer PB or Buffer PBI, then mix well.

2. Load the cDNA onto the MinElute column:

a. Load 600 µL of the sample containing Buffer PB or Buffer PBI onto the
MinElute column.
b. Spin the column at 13,000 × g for 1 minute.
c. Discard the flowthrough.

3. Wash the cDNA:

a. Return the MinElute column to the microcentrifuge tube.
b. Add 750 µL of Buffer PE to the MinElute column.
c. Spin the column at 13,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the MinElute column to the microcentrifuge tube.
f. Spin the column at 13,000 × g for 1 minute.

4. Elute the cDNA in a clean microcentrifuge tube:

a. Place the MinElute column in a clean microcentrifuge tube.
b. Add 10 µL of Buffer EB to the center of the MinElute column.
c. Wait 1 minute, then spin the column at 13,000 × g for 1 minute.

SOLiD® Total RNA-Seq Kit 75

 Appendix B Supplemental Information
B Gel size selection for Whole Transcriptome workflow standard input

Size select the Perform gel purification for precise size-selection and for the highest quality mapping.
cDNA Use Invitrogen Novex® pre-cast gel products, 50 bp DNA Ladder, and SYBR® Gold
nucleic acid gel stain:
• Invitrogen Novex® 6% TBE-Urea Gel 1.0 mM, 10 Well
• Invitrogen Novex® TBE Running Buffer (5✕)
• Invitrogen Novex® TBE-Urea Sample Buffer (2✕)
• Invitrogen XCell SureLock™ Mini-Cell
• Invitrogen 50 bp DNA Ladder
• Invitrogen SYBR® Gold nucleic acid gel stain
For more instructions on running Novex gels, refer to the Novex® Pre-Cast Gel
Electrophoresis Guide. For more instructions on staining the gel, refer to the SYBR® Gold
Nucleic Acid Gel Stain manual.

1. Prepare the gel as described in the Novex® Pre-Cast Gel Electrophoresis Guide:
a. Prepare 1000 mL of 1✕ TBE Running Buffer:

Component Volume

Novex® TBE Running Buffer (5✕) 200 mL

Deionized water 800 mL
Total volume 1000 mL

b. Place the Novex® 6% TBE-Urea Gel in the XCell SureLock™ Mini-Cell.

c. Use a marker to draw a line that is 1 cm below the middle of the gel, as
shown in the figure in step 6 on page 77.
d. Add 1✕ TBE Running Buffer to the Upper Buffer Chamber and the Lower
Buffer Chamber.

2. Dilute the 50 bp DNA Ladder:

Component Volume Concentration

50 bp DNA Ladder 1 µL 1 µg/µL

RNase-free water 24 µL –
Total volume 25 µL 40 ng/µL

76 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Gel size selection for Whole Transcriptome workflow standard input B

3. Prepare the cDNA and the DNA ladder:

a. Mix 5 µL of the cDNA with 5 µL of 2✕ Novex TBE-Urea Sample Buffer.
b. Mix 5 µL of the 40 ng/µL 50 bp DNA Ladder with 5 µL of 2✕ Novex TBE-
Urea Sample buffer.
c. Heat the cDNA and the DNA Ladder at 95°C for 3 minutes.
d. Snap-cool the tubes on ice. Leave the tubes on ice for less than 30 minutes.
Note: Do not leave denatured samples on ice for longer than 30 minutes. If
the denatured samples are left on ice for longer than 30 minutes, repeat
step 3c before loading the samples.

4. Immediately before you load each sample, flush the well of the gel several times
with 1X TBE Running Buffer to remove urea from the wells.
Note: Flushing the wells is important to obtain sharp bands.

5. Load the cDNA samples and the DNA Ladder.

Note: Follow these guidelines when loading the gel:
• Do not use the lanes next to the edges of the gel (lanes #1 and #10).
• Load the DNA Ladder on both sides of each cDNA sample to help you make
accurate cuts.
• While loading, place the pipette tip as close to the bottom of the well as
possible, and load the sample slowly. It is important to keep the sample
compact in the gel.

6. Run the gel at 180 V until the leading dye front is 1 cm below the middle of the gel
(~25 minutes).

Run the gel until the leading dye front is 1 cm

below the middle of the gel

IMPORTANT! Shortening or lengthening the running time results in inferior

resolution.

7. Add 5 µL of the SYBR Gold nucleic acid gel stain to 50 mL of 1✕ TBE Running
Buffer, then stain the gel for 5–10 minutes.

SOLiD® Total RNA-Seq Kit 77

 Appendix B Supplemental Information
B Gel size selection for Whole Transcriptome workflow standard input

8. Illuminate the stained gel, then excise the gel containing 150–250 nt cDNA:
Note: Be careful not to include extra gel that does not contain any cDNA.
a. Using a clean razor blade, make horizontal cuts to excise the gel containing
150–250 nt cDNA.

Make the horizontal cuts first to obtain

the desired insert length

Note: If you are using a UV transilluminator to visualize the reaction

products, work quickly to limit their exposure to UV radiation.
Note: To obtain the desired insert length, you can adjust the cuts. However,
too much gel will severely inhibit the PCR reactions. See Table 1 on page 79
for the expected lengths of the insert and PCR product according to the
length of the cDNA that is excised from the gel.
b. Reduce the width of the gel piece by making vertical cuts on both edges of
the smear.

Make the vertical cuts on both

edges of the smear

Note: The width of the smear is normally more narrow than the width of the
well in the gel. Make the cuts carefully to minimize the amount of extra gel in
the gel piece.

9. Transfer the gel piece to a clean working area, maintaining the orientation of the
gel, then cut the gel vertically into 4 pieces using a clean razor blade. Each gel slice
should be about 1 mm × 6 mm.

Cut the gel vertically into 4 pieces

78 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Gel size selection for Whole Transcriptome workflow standard input B

10. Place the two gel slices from the middle of the lane individually into clean 0.2-mL
PCR tubes, and place the outside gel slices into a clean 1.5-mL microcentrifuge
tube for storage.
To generate sufficient cDNA for accurate quantitation, you need to run 2
amplification reactions using 2 gel pieces, 1 gel piece in each reaction. You may
store the other 2 pieces for 2 weeks at –20°C.
Note: To maximize the yield for SOLiD System sequencing, use the 2 gel pieces
from the middle of the lane.

Example of 1st Figure 14 shows 5 µL of purified cDNA from HeLa poly(A) RNA run on a Novex 6%
round size TBE-Urea Gel with the Invitrogen 50 bp DNA Ladder. The white rectangle indicates
the area of the gel to excise. Each vertical slice can be used for one 100-µL PCR.
selection
Figure 14 Example of size selection of cDNA from HeLa poly(A) RNA

Region of excision

Expected lengths Table 1 Expected lengths of the insert and PCR according to excised cDNA length
of the insert and
Excised cDNA length (nt) Insert length (bp) PCR product length (bp)
PCR product
according to 50 ~0 ~100
excised cDNA 100 ~50 ~150
length 150 ~100 ~200
200 ~150 ~250
250 ~200 ~300

SOLiD® Total RNA-Seq Kit 79

 Appendix B Supplemental Information
B Gel size selection for Whole Transcriptome workflow standard input

Amplify the cDNA Use components from the SOLiD™ Total RNA-Seq Kit:
• Nuclease-free water
• 10x PCR buffer
• dNTP Mix
• SOLiD™ 5’ PCR Primer
• AmpliTaq® DNA Polymerase
• SOLiD™ 3’ PCR Primer

1. For each cDNA sample, prepare 95 µL PCR mix.

Component Volume (µL)

One100-µL reaction Two 100-µL reactions

Nuclease-free water 71.8 143.6

10x PCR Buffer 10.0 20.0
dNPT Buffer 8.0 16.0
SOLiD 5’ PCR Primer 2.0 4.0
AmpliTaq® DNA Polymerase 1.2 2.4
Total Volume 93.0 186.0

2. Transfer 5 µL of each cDNA sample into a new 0.2mL PCR tube.

3. Add 93 µL PCR mix to each cDNA sample.
4. Add 2 µL 3' PCR Primer to each sample.
Note: To prepare cDNA libraries for multiplex SOLiD™ System sequencing,
substitute with the barcoded SOLiD™ 3' PCR Primers from your SOLiD™ RNA
Barcoding Kit.

5. Run the PCR reactions in a thermal cycler:

Stage Temperature Time

Hold 95°C 5 minutes

95°C 30 seconds
Cycle (15 cycles) 62°C 30 seconds
72°C 30 seconds
Hold 72°C 7 minutes

Note: Run 15 cycles if you started with 50-100ng of fragmented RNA. If

necessary, adjust the numbers of cycles accordingly to the amount of input
fragmented RNA, but for optimal results run between 12 and 18 cycles. Too many
cycles results in overamplification and changes the expression profile.

80 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Gel size selection for Whole Transcriptome workflow standard input B

Purify the Use the Invitrogen PureLink® PCR Micro Kit:

amplified DNA • PureLink® Micro Kit Column
• Collection Tube
• Binding Buffer (B2)
• Wash Buffer (W1)
• PureLink® Elution Tube

IMPORTANT! Do not use other PCR purification kits. Other purification kits are not as
effective in the removal of unincorporated primers. Unincorporated primers can affect
the final quantitation and emulsion PCR.

1. Before using the PureLink® Micro Kit Column, place it in a clean Collection Tube,
then spin the column at 10,000 × g for 1 minute.

2. Prepare the sample:

a. Combine the two 100-µL PCR reactions in a new 1.5-mL tube.

IMPORTANT! If you used two different barcoded SOLiD 3′ Primers for your
sample, do not combine the PCRs at this step.

b. Add 800 µL of Binding Buffer (B2) to the tube, then mix well.

3. Load the sample onto the PureLink® Micro Kit Column:

a. Load 500 µL of the sample containing Binding Buffer (B2) onto the
PureLink® Micro Kit Column.
b. Spin the column at 10,000 × g for 1 minute.
c. Discard the flowthrough.
d. Load the remaining 500 µL of the sample containing Binding Buffer (B2)
onto the column.
e. Spin the column at 10,000 × g for 1 minute.
f. Discard the flowthrough.

4. Wash the DNA:

a. Return the column to the Collection Tube.
b. Add 600 µL of Wash Buffer (W1) to the column.
c. Spin the column at 10,000 × g for 1 minute.
d. Discard the flowthrough.
e. Return the column to the Collection Tube.
f. Spin the column at 14,000 × g for 1 minute.

5. Elute the DNA in a clean PureLink® Elution Tube:

a. Place the column in a clean PureLink® Elution Tube.
b. Add 10 µL of Elution Buffer to the center of the membrane.
c. Wait 1 minute, then spin the column at 14,000 × g for 1 minute.
d. Repeat step 5b through step 5c for a total elution volume of 20 µL.

SOLiD® Total RNA-Seq Kit 81

 Appendix B Supplemental Information
B Gel size selection for Whole Transcriptome workflow standard input

Assess the yield Use a NanoDrop spectrophotometer, and the Agilent 2100 Bioanalyzer with the DNA
and size 1000 Kit (Agilent).
distribution of the 1. Measure the concentration of the purified DNA with a NanoDrop®
amplified DNA spectrophotometer, and if necessary, dilute the DNA to <50 ng/µL for accurate
quantitation with the DNA 1000 Kit.

2. Run 1 µL of the purified DNA on an Agilent 2100 Bioanalyzer with the DNA 1000
Kit. Follow the manufacturer’s instructions for performing the assay.

3. Using the 2100 expert software, perform a smear analysis to quantify the
percentage of DNA that is 25–200 bp.

Percent of DNA in
Next steps
the 25–200 bp range

Less than 20% Proceed with SOLiD™ System templated bead

preparation.
Refer to the Applied Biosystems SOLiD™ 4 System
Templated Bead Preparation Guide (PN 4448378) or to
SOLiD™ EZ Bead Emulsifier Getting Started Guide (Part no.
4441486)
Greater than 20% Follow the troubleshooting instructions for “Normal yield
and bad size distribution in the amplified library” on
page 84.

Note: For instructions on how to perform the smear analysis, refer to the Agilent
2100 Bioanalyzer 2100 Expert User’s Guide by Agilent.

4. Determine the median peak size (bp) and molar concentration (nM) of the cDNA
library using the Agilent software. For more information, see “Determine the
median size” on page 89.
Note: The mass concentration of the cDNA must be <50 ng/µL for accurate
quantitation with the DNA 1000 Kit.

Alternatively, obtain the mass concentration by another method, and convert the
mass concentration to molar concentration.

82 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Gel size selection for Whole Transcriptome workflow standard input B

Typical size profiles Typical size distributions (Agilent 2100 Bioanalyzer profiles) of amplified libraries
of amplified prepared from HeLa poly(A) RNA (Figure 15) and rRNA-depleted HeLa RNA
(Figure 16) using the SOLiD™ Total RNA-Seq Kit are shown.
libraries
Figure 15 Size distribution of amplified library prepared from HeLa poly(A) RNA

Figure 16 Size distribution of amplified library prepared from rRNA-depleted HeLa total RNA

SOLiD® Total RNA-Seq Kit 83

 Appendix B Supplemental Information
B Troubleshooting

Figure 17 Size distribution of amplified library prepared from WT control total RNA

Troubleshooting

Observation Possible cause Solution

Low yield and no PCR products The gel ran too long or too much gel Reduce the running time (step 6 on
was added to the PCR page 77) and add less gel to the PCR.

Normal yield and bad size distribution Too much sample was loaded on the Decrease the volume of sample
in the amplified library Novex® TBE-Urea Gel loaded to less than 10 µL (step 5 on
page 77).
The wells of the Novex TBE-Urea Gel Before you load the samples, flush the
contained urea wells of the gel several times with 1X
TBE Running Buffer to remove urea
from the wells and to obtain sharp
bands (step 4 on page 77).
You decreased the volume of sample Fragmented RNA sample contains too Perform “Second-round size selection
loaded on the Novex® TBE-Urea Gel, many small fragments of amplified cDNA” on page 85.
but smear analysis of the purified
Size selection was not successful
amplified cDNA shows that >20% of
the cDNA is in the 25–200 bp range
Normal or high yield but the purified Nonspecific amplification Increase the PCR annealing
amplified cDNA shows one or more temperature to 68–72 °C.
sharp peaks between 100 and 150 bp
in the Agilent® 2100 Bioanalyzer™
Instrument trace

84 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Second-round size selection of amplified cDNA B

Second-round size selection of amplified cDNA

Perform a second round of size selection of the amplified, double-stranded cDNA
using nondenaturing polyacrylamide gel electrophoresis if:
• For whole transcriptome libraries: The percent of DNA that is 25–200 bp is greater
than 20% (from step 3 on page 18).
• For small RNA libraries: The ratio of 120–130-bp DNA: 25–150-bp DNA is less
than 50% (from step 2 on page 63)

Size-select the Use Invitrogen Novex® pre-cast gel products, 10 bp or 50 bp DNA Ladder, and SYBR®
amplified cDNA Gold nucleic acid gel stain:
• Invitrogen Novex® 6% TBE Gel 1.0 mM, 10 Well
• Invitrogen Novex® TBE Running Buffer (5✕)
• Ambion® Gel Loading Solution
• Invitrogen Novex® TBE Sample Buffer (2✕)
• Invitrogen XCell SureLock™ Mini-Cell
• Invitrogen 50 bp DNA Ladder for whole transcriptome libraries or the 10 bp
DNA Ladder for small RNA libraries
• Invitrogen SYBR® Gold nucleic acid gel stain
For more instructions on running Novex gels, refer to the Novex® Pre-Cast Gel
Electrophoresis Guide.
For more instructions on staining the gel, refer to the SYBR® Gold Nucleic Acid Gel Stain
manual.

1. Add Gel Loading Solution (AM8556) to the eluted cDNA:

• For whole transcriptome libraries: Add 4 µL Gel Loading Solution to the
eluted cDNA (~20 µL) from step 5 on page 81.
• For small RNA libraries: Add 2 µL Gel Loading Solution to the eluted cDNA
(~10 µL) from step 4 on page 57.

IMPORTANT! Do not heat the samples before loading.

2. Prepare the gel as described in the Novex® Pre-Cast Gel Electrophoresis Guide:
a. Prepare 1000 mL of 1✕ TBE Running Buffer using Novex® TBE Running
Buffer (5✕).

Component Volume

Novex® TBE Running Buffer (5✕) 200 mL

Deionized water 800 mL
Total volume 1000 mL

b. Place the Novex® 6% TBE Gel in the XCell SureLock™ Mini-Cell.

c. Add 1✕ TBE Running Buffer to the Upper Buffer Chamber and the Lower
Buffer Chamber.

SOLiD® Total RNA-Seq Kit 85

 Appendix B Supplemental Information
B Second-round size selection of amplified cDNA

3. Load the sample on the Novex 6% TBE Gel:

• For whole transcriptome libraries: Load the sample into 3 adjacent wells
(8 µL per well) and include a separate well for 5 µL 50 bp DNA Ladder
(40 ng/µL)
• For small RNA libraries: Load the sample into 2 adjacent wells (6 µL per
well) and include a separate well for 5 µL 10 bp DNA Ladder (40 ng/µL)
Note: Include a separate well with 5 µL 50 bp Ladder (40 ng/µL).

4. Run the gel at ~140 V for ~45 minutes, or until the front dye reaches the bottom of
the gel.
Note: Nondenaturing gels must be run slowly to avoid heat denaturation of the
samples.

5. Stain the gel with SYBR® Gold dye, following the manufacturer’s instructions.
6. Illuminate the stained gel, then excise the gel in the appropriate size range for
your library:
• For whole transcriptome libraries: 200–300 bp
• For small RNA libraries: 110–130 bp
Note: If you are using a UV transilluminator to visualize the nucleic acid, work
quickly to limit its exposure to UV radiation.

86 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Second-round size selection of amplified cDNA B

Purify the Use PAGE Elution Buffer (recipe below) and Ambion® Spin Columns and Tubes
amplified cDNA (AM10065).
from the gel 1. Prepare ~600 µL PAGE Elution Buffer for each sample.

Component Volume (mL)

TE Buffer, pH 8 (10 mM Tris-HCl, ph 8, 1 mM EDTA) 5
5 M ammonium acetate (2.5 M final concentration) 5
Final volume 10

2. Shred the gel piece:

a. Use a 21-gauge needle to puncture through the bottom-center of a 0.5-mL
microcentrifuge tube.
b. Place the gel piece in the punctured 0.5-mL tube, then place the 0.5-mL tube
into a larger, 1.5-mL, nuclease-free microcentrifuge tube.
c. Spin for 3 minutes at 13,000 x g to shred the gel.
d. Place the 1.5-mL tube containing the shredded gel piece on ice.
e. Inspect the 0.5-mL tube, and if any gel pieces remain, repeat the
centrifugation step into a fresh 1.5-mL tube. Pool the gel pieces into one
collection tube using a pipette tip.

3. Elute the DNA in PAGE elution buffer:

a. Add 300 µL of PAGE Elution Buffer to the shredded gel pieces.
b. Incubate the mixture overnight at room temperature, with gentle agitation.
c. Transfer the buffer, which contains eluted DNA, to a fresh tube, leaving the
gel fragments behind.
Store the DNA on ice during the second elution (step 3e).
d. Add another 300 µL of PAGE Elution Buffer to the shredded gel pieces.
e. Incubate the buffer and gel pieces for 1 to 2 hours at 37°C, with gentle
agitation.

4. Remove the gel pieces from the sample using a filter spin column:
a. Combine the PAGE elution buffer from step 3c with the buffer plus gel slurry
from step 3e.
b. Cut a pipette tip to make a larger opening and use it to transfer the combined
PAGE elution buffer and gel slurry from each sample to a Spin Column.
c. Spin the Spin Column at top speed for 5 minutes to remove gel pieces. The
DNA is now in the flowthrough.

Alternatively, you can use a 0.45 µm-filter spin column from another
manufacturer for this step, following the manufacturer’s instructions for the
maximum centrifugation speed.

SOLiD® Total RNA-Seq Kit 87

 Appendix B Supplemental Information
B Using 2100 expert software to assess whole transcriptome libraries

5. Precipitate the DNA, then resuspend in 20 µL of Nuclease-free Water:

a. Add 1/100 volume of glycogen and 0.7 volume of isopropanol to each
sample.
b. Mix thoroughly, then incubate at room temperature for 5 minutes.
c. Spin the sample at 13,000 x g for 20 minutes at room temperature.
d. Carefully remove and discard the supernatant, then air dry the pellet.
e. Resuspend the DNA pellet in 20 µL of Nuclease-free Water.
Note: Accurate quantitation of the DNA is important for the downstream
SOLiD™ System emulsion PCR titration step. The resuspension volume
should yield DNA sufficiently concentrated for accurate measurements
(~10 ng/µL).

Using 2100 expert software to assess whole transcriptome

libraries
Perform a smear Perform a smear analysis to quantify the percentage of DNA in the 25–200 bp size
analysis range.

1. In the 2100 expert software, select ViewSetpoints.

2. On the Global tab, select Advanced settings.

3. In the Sample Setpoints section of the Advanced settings, select the Perform
Smear Analysis checkbox, then double-click Table.

88 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Using 2100 expert software to assess whole transcriptome libraries B

4. Set the smear regions in the Smear Regions dialog box:

a. Click Add, then enter 25 bp and 200 bp for the lower and upper limits,
respectively.
These settings are used to determine the percentage of total product that is
25–200 bp in length.
b. Click Add, enter 25 bp and 225 bp, then click OK.
This is an arbitrary upper limit which is used to determine the median size.

5. Select the Region Table tab.

6. In the Region Table, review the percentage of the total product in the size ranges
you set.

Determine the On the Region Table tab, drag the upper limit line that you set in step b on page 89 to
median size the left or right until the Region Table indicates 50% of Total.

SOLiD® Total RNA-Seq Kit 89

 Appendix B Supplemental Information
B Using 2100 expert software to assess whole transcriptome libraries

Analyze multiple On the Peak Table tab, you may observe that the bioanalyzer software identified
peaks as one peak multiple peaks in a region that you want to consider as one peak. To obtain one
concentration and automatically determine median size for a peak region, manually
set the size range of the desired peak region.

1. In the bottom left corner of the software window, select the Peak Table tab.

2. Right-click anywhere on the electropherogram, then select Manual Integration.

3. To remove multiple peaks:

a. Place the cursor on the peak to remove, right-click, then select Remove Peak.

b. Repeat until one peak remains within the region of interest

90 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Small RNA enrichment B

c. Drag the lower and upper region limits of the region until the entire library
is included.

Drag the lower region Drag the upper region

limit toward the left limit toward the right

4. The software recalculates the median size (bp), concentration (ng/µL), and
molarity (nM) of the peak region and displays the values in the Peak Table.

Small RNA enrichment

If your RNA sample contains < 0.5% miRNA, perform small RNA enrichment using
the Invitrogen PureLink® miRNA Isolation Kit using this purification procedure. If
you are using the PureLink® miRNA Isolation Kit to isolate small RNA from cells or
tissue, refer to the PureLink® miRNA Isolation Kit Instruction Manual.

1. Prepare the sample:

a. Resuspend 5–50 µg total RNA in 90 µL Nuclease-free Water.
b. Add 300 µL Binding Buffer (L3) and 210 µL 100% ethanol, then vortex to mix
well.

2. Load the total RNA onto a Spin Cartridge:

a. Load 600 µL of the sample containing Binding Buffer and ethanol onto the
Spin Cartridge in a Collection Tube.

SOLiD® Total RNA-Seq Kit 91

 Appendix B Supplemental Information
B Small RNA enrichment

b. Spin the cartridge at 12,000 × g for 1 minute.

IMPORTANT! Total RNA is bound to the cartridge and small RNA is in the
flowthrough. Keep the flowthrough.

c. Transfer the flowthrough to a new 1.5-mL tube with a lid.

d. Discard the cartridge.

3. Add 700 µL 100% ethanol to the flowthrough, then vortex to mix well.
4. Load the small RNA onto a second Spin Cartridge:
a. Load 700 µL of the sample containing 100% ethanol onto a second Spin
Cartridge in a Collection Tube.
b. Spin the cartridge at 12,000 × g for 1 minute.
c. Discard the flowthrough.
d. Return the cartridge to the collection tube.
e. Load the remaining 700 µL of the sample containing 100% ethanol onto the
cartridge.
f. Spin the cartridge at 12,000 × g for 1 minute.
g. Discard the flowthrough.

5. Wash the small RNA:

a. Return the cartridge to the collection tube.
b. Add 500 µL of Wash Buffer (W5) with ethanol to the Spin Cartridge.
c. Spin the cartridge at 12,000 × g for 1 minute.
d. Discard the flowthrough.

6. Wash the small RNA a second time:

a. Return the cartridge to the collection tube.
b. Add 500 µL of Wash Buffer (W5) with ethanol to the Spin Cartridge.
c. Spin the cartridge at 12,000 × g for 1 minute.
d. Discard the flowthrough and the collection tube.

7. Remove any residual Wash Buffer:

a. Place the Spin Cartridge in a Wash Tube supplied with the kit.
b. Spin the cartridge at 16,000 × g for 3 minutes.
c. Discard the flowthrough and the Wash Tube.

8. Elute the small RNA:

a. Place the Spin Cartridge in a clean Recovery Tube supplied with the kit.
b. Add 50 µL of Sterile, RNase-free Water to the center of the Spin Cartridge.
c. Incubate the Spin Cartridge at room temperature for 1 minute.
d. Spin the cartridge at 16,000 × g for 1 minute.

Store the small RNA at –80°C or assess the quality and quantity of the small RNA-
enriched sample (page 54).

92 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Using 2100 expert software to assess small RNA libraries B

Using 2100 expert software to assess small RNA libraries

Review the median The 2100 expert software automatically calculates the median size (bp) of miRNA
size ligation products.
Select the Peak Table tab, then review the median size in the Peak Table and at the top
of the peak in the electropherogram. The median size should be ~122–125 bp.

Perform a smear Perform a smear analysis to quantify the percentage of DNA in the 25–150 bp and 120–
analysis 130 bp size range. The desired size range for miRNA ligation products is 120–130 bp.

1. In the 2100 expert software, select ViewSetpoints.

2. On the Global tab, select Advanced settings.

3. In the Sample Setpoints section of the Advanced settings, select the Perform
Smear Analysis checkbox, then double-click Table.

SOLiD® Total RNA-Seq Kit 93

 Appendix B Supplemental Information
B Using 2100 expert software to assess small RNA libraries

4. Set the smear regions in the Smear Regions dialog box:

a. Click Add, then enter 25 bp and 150 bp for the lower and upper limits,
respectively.
b. Click Add, enter 120 bp and 130 bp, then click OK.

5. Select the Region Table tab.

6. In the Region Table, review the area values for each of the size ranges you set.

94 SOLiD® Total RNA-Seq Kit

 Appendix B Supplemental Information
Using 2100 expert software to assess small RNA libraries B

Determine the % Using the area values from the Region Table, calculate the % miRNA library in the 120–
miRNA library 130 bp region as a fraction of the 25–150 bp region using the formula:

% miRNA library =
( Area from 120–130 bp

Area from 25–150 bp ) x 100

If the % miRNA library is less than 50%, we strongly recommend that you perform a
second-round size selection using a nondenaturing polyacrylamide gel, such as the
Novex 6% TBE Gel (page 85).

Example % miRNA library calculation

In the example below, the % miRNA library is 73%.

% miRNA library =
( ) 67.3
92.0
x 100 = 73%

SOLiD® Total RNA-Seq Kit 95

 Appendix B Supplemental Information
B Using 2100 expert software to assess small RNA libraries

96 SOLiD® Total RNA-Seq Kit

 C PCR Good Laboratory Practices

When preparing samples for PCR amplification:

• Use a positive-displacement pipette or aerosol-resistant pipette tips.
• Follow proper pipette-dispensing techniques to prevent aerosols.
• Wear clean gloves and a clean lab coat (not previously worn while handling
amplified PCR products or used during sample preparation).
• Change gloves whenever you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for:
– Sample preparation
– PCR setup
– PCR amplification
– Analysis of PCR products
• Never bring amplified PCR products into the PCR setup area.
• Open and close all sample tubes carefully. Centrifuge tubes before opening. Try
not to splash or spray PCR samples.
• Keep reactions and components capped as much as possible.
• Clean lab benches and equipment periodically with 10% bleach solution. Use
DNAZap™ Solution (PN AM9890).

SOLiD® Total RNA-Seq Kit 97

 Appendix C PCR Good Laboratory Practices
C

98 SOLiD® Total RNA-Seq Kit

 D Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified

in the user documentation may result in personal injury or damage to the
instrument or device. Ensure that anyone using this product has received
instructions in general safety practices for laboratories and the safety
information provided in this document.
• Before using an instrument or device, read and understand the safety
information provided in the user documentation provided by the
manufacturer of the instrument or device.
• Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.

SOLiD® Total RNA-Seq Kit 99

 Appendix D Safety
D Chemical safety

Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,

ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below, and consult the relevant
SDS for specific precautions and instructions:
• Read and understand the Safety Data Sheets (SDSs) provided by the
chemical manufacturer before you store, handle, or work with any chemicals
or hazardous materials. To obtain SDSs, see the “Documentation and
Support” section in this document.
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
• Minimize the inhalation of chemicals. Do not leave chemical containers
open. Use only with adequate ventilation (for example, fume hood).
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
the manufacturer's cleanup procedures as recommended in the SDS.
• Handle chemical wastes in a fume hood.
• Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
• After emptying a waste container, seal it with the cap provided.
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.
• IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.

100 SOLiD® Total RNA-Seq Kit

 Appendix D Safety
Biological hazard safety D

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,

infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective equipment, which includes
but is not limited to: protective eyewear, face shield, clothing/lab coat, and
gloves. All work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in
the following:
In the U.S.:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories found at:
[Link]/biosafety
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030), found at: [Link]/nara/cfr/waisidx_01/
29cfr1910a_01.html
• Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
• Additional information about biohazard guidelines is available at:
[Link]
In the EU:
Check local guidelines and legislation on biohazard and biosafety precaution
and refer to the best practices published in the World Health Organization
(WHO) Laboratory Biosafety Manual, third edition, found at: [Link]/
csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/

SOLiD® Total RNA-Seq Kit 101

 Appendix D Safety
D Biological hazard safety

102 SOLiD® Total RNA-Seq Kit

 Documentation and Support

Obtaining SDSs
Safety Data Sheets (SDSs) are available from:
• [Link]/sds
or
• [Link]/sds
Note: For the SDSs of chemicals not distributed by Life Technologies, contact the
chemical manufacturer.

Obtaining support
For the latest services and support information for all locations, go to:
• [Link]
or
• [Link]
At the website, you can:
• Access worldwide telephone and fax numbers to contact Technical Support and
Sales facilities
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Search for user documents, SDSs, vector maps and sequences, application notes,
formulations, handbooks, certificates of analysis, citations, and other product
support documents
• Obtain information about customer training
• Download software updates and patches

Kit documentation
The following documents are available for the SOLiD™ Total RNA-Seq Kit:

SOLiD® Total RNA-Seq Kit 103

Documentation and Support
Related documentation

Part
Document Description
number

SOLiD™ Total RNA-Seq 4452437 Provides product information, step-by-step

Kit Protocol instructions for using the SOLiD Total RNA-Seq
Kit, troubleshooting information, ordering
information, and supplemental information.
SOLiD™ Total RNA-Seq 4452438 Provides abbreviated instructions for using the
Kit for Whole SOLiD Total RNA-Seq Kit to create an amplified
Transcriptome Libraries cDNA library of the whole transcriptome.
Quick Reference Card
SOLiD™ Total RNA-Seq 4452439 Provides abbreviated instructions for using the
Kit for Small RNA SOLiD Total RNA-Seq Kit to create an amplified
Libraries Quick Reference cDNA library of the small RNAs.
Card

Related documentation
When using this protocol, you may find the documents listed below useful. To obtain
this and additional documentation, see “Obtaining support” on page 103.

Document Part number

Applied Biosystems SOLiD™ 4 System SETS Software User Guide 4448411

Applied Biosystems SOLiD™ 4 System Templated Bead Preparation Guide 4448378
SOLiD™ EZ Bead Emulsifier Getting Started Guide 4441486
SOLiD™ EZ Bead™ Amplifier Getting Started Guide 4443494

104 SOLiD® Total RNA-Seq Kit

 Bibliography

Tissot, C. (Agilent® Technologies), 2008. Analysis of miRNA content in total RNA

preparations using the Agilent® 2100 Bioanalyzer™. 8 pp. Publication Number 5989-
7870EN.

SOLiD® Total RNA-Seq Kit 105

Headquarters
5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288
For support visit [Link]/support
[Link]