RNA library preparation and sequencing: Processing order was randomized prior to ribosomal RNA depletion, and samples were processed in batches of 8. In order to expedite sequencing, processing began before extraction was complete and randomization occurred among all available extracted samples in sets of 120 to 226. Briefly, rRNA was depleted from about 1 ug of total RNA using Ribozero Magnetic Gold kit (Illumina/Epicenter Cat #MRZG12324) to enrich polyadenylated coding RNA and non-coding RNA. The Pitt case/control pairs were batched together in each processing step, including Ribozero depletion, sequence library preparation and sequencing lane. The sequencing library was prepared using the TruSeq RNA Sample Preparation Kit v2 (RS-122-2001-48 reactions) in batches of 24 samples. The insert size and DNA concentration of the sequencing library was determined on Agilent Bioanalyzer and Qubit, respectively. A pool of 10 barcoded libraries were layered on a random selection of two of the eight lanes of the Illumina flow cell at appropriate concentration and bridge amplified to ~250 million raw clusters. One-hundred base pair paired end reads were obtained on a HiSeq 2500. The sequencing
