RNA-Seq Analysis and Pathway Enrichment

RNA was extracted using Trizol (Invitrogen). RNA samples were sent to the Genomics Research Center of the University of Rochester for RNA-Seq. The TruSeq RNA Sample Preparation Kit V2 (Illumina) was used for library construction. The libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina) at a concentration of 8 pM per lane. Single end reads of 100 nt were generated for each sample. The sequencing was performed using the Illumina high-throughput HiSeqTM 2500. Normalization and differential expression analysis were carried out using DESeq2^{46 (link)}. Ontology and pathway analyses were performed using Enrichr program^{26 (link)}. TFBS enrichment analysis was done using oPOSSUM program^{27 (link)}. The parameters used in the oPOSSUM were as follows: conservation cut-off = 0.4, matrix score threshold = 85%, analyzed sequences = 2 kb upstream of transcription start sites, Z-score cut-off = 10, Fisher score cut-off = 7.