RNA-Seq and miRNA-Seq Analysis Protocol

RNA sequencing analysis was performed as described [16 ]. Sequencing RNA libraries were prepared according to the manufacturer’s protocol (TruSeq RNA Sample Prep Kit v2, Illumina) and loaded onto flow cells (8-10pM) to generate cluster densities of 700,000/mm² following the standard protocol for the Illumina cBot and cBot Paired-end cluster kit version-3. Cells were sequenced on an Illumina HiSeq 2000 using TruSeq SBS kit version 3 and HCS v2.0.12 data collection software and data analyzed using the MAPRSeq v.1.2.1 system and the Bioinformatics Core standard tool, which includes alignment with TopHat 2.0.6 [17 (link), 18 (link)] and gene counts with the featureCounts software [19 (link)]. miRNA-Seq data were analyzed using CAP-miRSeq v1.1 [20 (link)] and normalization and differential expression analysis performed using edgeR 2.6.2 [21 (link)]. Gene expression was normalized to 1 million reads and corrected for gene length (reads per kilobasepair per million mapped reads, RPKM), and miRNA expression levels expressed as normalized total reads.