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RNA-seq Tutorial: Mouse Embryo Insights

This tutorial provides a hands-on approach to processing and interpreting RNA-seq data from early mouse embryos (GSE45719) to explore genomic and epigenetic regulation during reproduction and development. It outlines the steps for data preparation, downloading FASTQ files, quality control, and alignment using STAR and featureCounts. The dataset includes multiple single-cell samples across various developmental stages, facilitating the analysis of gene expression changes during preimplantation development.

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4 views2 pages

RNA-seq Tutorial: Mouse Embryo Insights

This tutorial provides a hands-on approach to processing and interpreting RNA-seq data from early mouse embryos (GSE45719) to explore genomic and epigenetic regulation during reproduction and development. It outlines the steps for data preparation, downloading FASTQ files, quality control, and alignment using STAR and featureCounts. The dataset includes multiple single-cell samples across various developmental stages, facilitating the analysis of gene expression changes during preimplantation development.

Uploaded by

minhnhut.lxag1988
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd

Tutorial: From RNA-seq to Genomic &

Epigenetic Insights — Reproduction &


Development (Using GSE45719 Data)
Goal: a hands-on tutorial demonstrating how to process and interpret RNA-seq data from early
mouse embryos (GSE45719) to uncover genomic and epigenetic regulation of reproduction and
development.

This version uses GSE45719, a well-annotated dataset with explicit stage information: zygote, 2-
cell, 8-cell, and blastocyst.

0. Overview & Biological Context


Biological question: How does gene expression change during mouse preimplantation
development, and how might genomic and epigenetic mechanisms regulate these transitions?

Dataset summary (GSE45719):

 Organism: Mus musculus


 Stages: Zygote, Early 2-cell, 8-cell, Early Blastocyst
 Platform: Illumina (single-end reads, Smart-Seq)
 Source: GEO → SRA → FASTQ (public domain)

Each stage has multiple single-cell samples with explicit SRR identifiers.

1. Sample Metadata & Preparation


Create a working directory:

mkdir -p data/fastq ref align counts results qc

Prepare a [Link] file:

sample stage fastq


zyg_1 zygote [Link]
zyg_2 zygote [Link]
2c_1 early_2-cell [Link]
2c_2 early_2-cell [Link]
8c_1 8-cell [Link]
8c_2 8-cell [Link]
bs_1 early_blastocyst [Link]
bs_2 early_blastocyst [Link]

2. Downloading FASTQ files from SRA


Use the SRA Toolkit to fetch the raw reads:

for SRR in SRR805451 SRR805452 SRR805283 SRR805284 SRR805241 SRR805242


SRR805294 SRR805295
do
prefetch $SRR
fasterq-dump $SRR
gzip ${SRR}.fastq
done

These are single-end reads; no need for --split-files.

3. Quality Control (FastQC + MultiQC)


fastqc -o qc/raw/ data/fastq/*.[Link]
multiqc -o qc/raw/ qc/raw/

Inspect per-base quality, adapter content, and duplication levels. Embryo single-cell RNA-seq
often has high duplication and variable coverage.

4. Alignment / Quantification
We use STAR for alignment and featureCounts for quantification.

# Build STAR index (once)


STAR --runThreadN 8 --runMode genomeGenerate \
--genomeDir ref/STAR_index \
--genomeFastaFiles ref/[Link] \
--sjdbGTFfile ref/[Link] \
--sjdbOverhang 100

# Align each sample


for sample in $(cut -f1 [Link] | tail -n +2)
do

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