RNA Sequencing Protocol for Transcriptomic Analysis

Complete methods for RNA sequencing are detailed in Thibodeau, et al.[33 (link)] RNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA) according to the manufacturer’s instruction. One sample failed library prep and was excluded from the study. Samples were sequenced on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3. A minimum of 50 million total reads per sample was required for analysis; 234 samples with <50 million total reads were re-sequenced and BAM files were merged if no quality issues were identified. RNA-seq data were analyzed using MAP-R-Seq pipeline, an integrated suite of open-source bioinformatics tools, along with in-house developed methods.[58 (link)] Gene counts were quantified for 55,601 genes based on ENSEMBL gene annotation. For genes mapping to both chromosomes X and Y, only the chromosome X version was retained.