Total RNA was evaluated for its quantity and quality using an Agilent Bioanalyzer 2100; RNA integrity numbers were all greater than 9. Fifty nanograms of total RNA was used for each sequencing library. cDNA library preparation was carried out at the Center for Medical Genomics, and included mRNA enrichment using oligo dT-beads to capture polyA RNA, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the KAPA mRNA Hyper Prep Kit Technical Data Sheet, KR1352 – v4.17 (Roche Corporate). Each resulting indexed library was quantified and its quality accessed by Qubit and Agilent Bioanalyzer, and all libraries were pooled at equal molarity. Five microliter aliquots of 2 nM pooled libraries per lane were denatured, neutralized and applied to flow cell deposition on both surfaces of the flow cell; library cluster amplification was performed on a cBot 2 System (Illumina, Inc.). The resulting flow cell was sequenced on an Illumina HiSeq 4000, 75 base paired-end sequencing (Illumina, Inc.). Approximately 30 M reads per library were generated (minimum 26.5 M, maximum 35.4 M). A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).
