RNA-seq Analysis of Mouse Transcriptome

Following RNA isolation (described above), RNA quality was assessed with a Bioanalyzer (Agilent) and the library was constructed with 1 μg total RNA and Illumina TruSeq Stranded Total RNA Ribo-Zero H/M/R Gold (Illumina, RS-122–2301) according to the manufacturer’s instructions. Libraries were quantified with a Bioanalyzer (Agilent) and Qubit 2.0 and sequenced on an Illumina NextSeq 500 instrument to a depth of at least 18 million reads, a 75-bp single read. The reads were mapped to the mouse genome with mm10 reference assembly from UCSC (https://genome.ucsc.edu/), using STAR (27 (link)). Reads mapped to genes were counted with HTSeq-count (28 (link)), using the union–intersection mode and refSeq mm10 transcriptome annotations from UCSC. Duplicate reads mapping to the same location were removed with Picard (http://broadinstitute.github.io/picard). Differential expression analysis was performed with edgeR (24 (link)). A gene was considered differentially expressed when the absolute fold change was above 2 and FDR < 0.05. A PCA plot was generated on the regularized log₂ transformed (rlog₂) read count data for all the samples. rlog₂ was performed with the DESeq2 package (29 (link)) of Bioconductor, and the PCA plot was generated with the ggplot2 package.