Processing order was randomized prior to ribosomal RNA depletion, and samples were processed in batches of 9 or 8. 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 sequencing library was prepared using the TruSeq RNA Sample Preparation Kit v2 (RS-122-2001-48 reactions) in batches of 17 samples. The insert size and DNA concentration of the sequencing library was determined on Agilent Bioanalyzer and Qubit, respectively. A pool of 8 or 9 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 data were simultaneously transferred (in real time) to storage computer cluster and then transferred to high performance computer cluster. The sequence data was processed for primary analysis to generate QC values. All data provided passed all of the following QC filters: samples were required to have a minimum of 50 million total reads and less than 5% rRNA alignment.
