RNA-seq Analysis of Mouse Transcriptome

10 ng of mRNA was used to make barcoded libraries using the Ion Total RNA-seq Kit V2 (Life Technologies). Resulting libraries were quantified and pooled to 75 pM before loading onto Ion P1 semiconductor chips using an Ion Chef and sequenced on an Ion Proton sequencer. Reads were mapped to the Mus musculus genome v10 and expression values were calculated using a Bowtie/TopHat/Cufflinks pipeline incorporated into the Ion Torrent Suite v.5.0.5 (Langmead, Trapnell, Pop, & Salzberg, 2009 (link); Trapnell et al., 2012 (link)). PCA analysis was conducted using the R package FactoMineR (Lê, Josse, & Husson, 2008 (link)) and differential gene expression was determined using the R DESeq2 package (Love, Huber, & Anders, 2014 (link)). Differentially expressed genes were defined as those that had a log₂ fold-change ≥ 1 and FDR ≤ 0.05. Statistical significance of expression differences for transcripts displayed in Figures 4 and 5 were evaluated using a one-way ANOVA followed by a Tukey's multiple comparison HSD test. Adjusted p-values less than 0.05 were considered significant.