RNA-seq reads were mapped to the hg38 reference genome using STAR (Dobin et al., 2013) aligner and mapped to Gencode version 24 gene annotations using RSEM(Li and Dewey, 2011). Genes with expression (< 1 FPKM) across all samples were filtered from all subsequent analysis. Differential gene expression analysis was performed on TMM normalized counts with EdgeR (Robinson et al., 2010). Multiple biological replicates were used for all comparative analysis. A p-value ≤ 0.001 and a 2-fold change in expression were used in determining significant differentially expressed genes for respective comparisons. PCA analysis was performed using the R package “rgl” and plotted using “plot3d”. Clustering was performed using R “hclust2” and visualized using Java Tree View 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). Differential gene analysis between groups was performed using the R package “limma” and significant genes (adjusted p < 0.01) were used for Gene ontology and pathway analysis. Gene ontology and pathway analysis was performed using Enrichr data base (http://amp.pharm.mssm.edu/Enrichr/) (Chen et al., 2013; Kuleshov et al., 2016).
