RNA-seq Data Processing and Analysis

Extracted RNA was controlled for integrity using the Agilent Bioanalyzer (RNA Analysis), Stranded libraries were prepared using the NEB mRNA stranded library prep Kit (New England Biolabs) and sequenced using HiSeq2500 sequencers (Illumina). Raw reads are clipped for adapter sequence, trimmed for minimum quality (Q30) in 3’ and filtered for minimum length of 32 bp using Trimmomatic.^{44 (link)} Surviving read pairs were aligned to Mus_musculus assembly GRCm38 by the ultrafast universal RNAseq aligner STAR^{45 (link)} using the recommended two passes approach. Aligned RNA Seq reads were assembled into transcripts and their relative abundance was estimated using Cufflinks and Cuffdiff.^{46 (link)} Exploratory analysis was conducted using various functions and packages from R and the Bioconductor project. Differential expression was conducted using both edgeR and DEseq. Terms from the Gene Ontology were tested for enrichment with the GOseq^{47 (link)} R package.