RNA-Seq Analysis of Transcriptome

Total RNA was isolated from duplicate wells of indicated conditions. RNA integrity and concentration were assessed using a BioAnalyzer (Agilent). RNA sequencing libraries were generated using Illumina mRNA TruSeq kit with dual index barcoding. Multiplexed libraries were sequenced at the Cold Spring Harbor Labs core sequencing facility. Approximately 8 million paired-end 76 bp reads were sequenced per replicate on a HiSeq 2500 instrument on RAPID mode. After removing adaptor sequences with Trimmomatic^{47 (link)}, RNA-Seq reads were aligned to GRCm38 – mm10 with STAR^{48 (link)}. Genome wide transcript counting was performed by HTSeq or featureCounts to generate FPKM matrix^{49 (link),50 (link)}. Differential expression analysis was performed with DESeq2 package in R^{51 (link)}. Genes with a real adjusted p value were used for downstream analyses.