RNA seq 流程
本文有待完善，请等待后续更新
Wu et al. 2022
- All the MeRIP–seq, RNA-seq and ATAC–seq data generated in this study are summarized in 41556_2022_915_MOESM2_ESM.xls.
- Index of /gsa/CRA003985/ (cncb.ac.cn)
DEGs While Inhibiting m6A
Plots:
- Fig. 5f: Volcano Plot A large number of genes were differentially expressed, between the DMSO and STM2457 treatment groups at the 4C and morula stages, but the gene expression of 2C embryos was almost unaffected
- Extended Data Fig. 8g: Principal component analysis (PCA) for embryos with DMSO and STM2457 treatment
- Supplementary Table 6
1 下载原始测序数据
1.1 下载数据
wget -r -c ftp://download.big.ac.cn/gsa/CRA003985/CRR355849/
1.2 检查数据的完整性
2 质量控制
2.1 质量控制
multiqc fastQC/ --outdir multiqc
2.2 接头修剪
trim_galore --cores 8 --output_dir ~/rnaSeqExercise/trimmed --basename basename --paired fq1 fq2
--paired参数代表双端测序--output参数指明输出目录--cores参数指明使用服务器内核数--basename参数重命名输出文件
# 测试输入
trim_galore --cores 8 --output_dir ~/rnaSeqExercise/trimmed \
--paired rawData/CRR355849/CRR355849_f1.fastq.gz rawData/CRR355849/CRR355849_r2.fastq.gz
2.3 接头修剪后的质量控制
fastqc ~/rnaSeqExercise/trimmed/*.fq.gz -t 8 -o ~/rnaSeqExercise/trimmedFastQC
multiqc ~/rnaSeqExercise/trimmedFastQC --outdir ~/rnaSeqExercise/trimmedMultiqc/
在 multiQC 结果的 Adapter Content 一项中，trim 后显示：
前后两次 fastQC 最显著的差异就在 Adapter Content 这一项中（左为前，右为后）
3 比对到参考基因组
3.1 参考基因组建立索引
MGSCv37 - mm9 - Genome - Assembly - NCBI (nih.gov)
Index of /goldenPath/mm9/bigZips (ucsc.edu)
hisat2-build -p 6 -f mm9.fa mm9
3.2 比对
hisat2 -p 15 --dta-cufflinks --no-mixed --no-discordant -x <mm9> -1 <fastq_file1> -2 <fastq_file2> | samtools view -@15 -Sb > <bam alignment file>
Hisat2的使用说明 - 简书 (jianshu.com)
3.3 对 Bam 文件进行质量控制
bamCoverage — deepTools 3.5.0 documentation
3.4 Bam 文件排序
samtools sort -@16 -o $bamSortedFile $bamFile
samtools flagstat <bamfilepath> > <flagstatfilepath>
3.5 计数
out <- featureCounts(files=idfpath, annot.ext = annopath,
isPairedEnd = TRUE, useMetaFeatures = TRUE,
isGTF = TRUE, GTF.featureType = "exon",
GTF.attrType = "gene_id",
GTF.attrType.extra = c("gene_name","gene_type"),
allowMultiOverlap = TRUE, fracOverlap = 0.5,
nthreads = 15)
