Total RNA Extraction and RNA-Seq Library Preparation

Total RNA was extracted from normal control and tumor tissues using the mirVana RNA purification kit (Thermo Fisher, Waltham, MA, USA) following manufacturer’s instructions. We initially evaluated RNA quality with NanoDrop Model 2000 spectrophotometer and used a 260 nm/280 nm absorbance ratio of ~2.0. RNAs with an RNA integrity number (RIN)^{26 (link)} of ≥7.0 via Trinean DropSense 16 spectrophotometer and Agilent Model 2100 bioanalyzer were then used for RNA sequencing. Messenger RNA-focused sequencing libraries were constructed using 500 ng of total RNA per sample that was converted to cDNA and linked to specific adaptors for sequencing via the Illumina TruSeq RNA library prep kit (Illiumina, San Diego, CA, USA). Indexed libraries for sequencing were re-concentrated into equimolar aliquots using the Agilent Model 2100 bioanalyzer and validated via an Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). The Illumina HiSeq. 4000 genome sequencing platform employing 150 bp paired-end sequencing by synthesis chemistry was used. All library preparation and sequencing procedures were performed in the Genome Core Facility of the University of Iowa Institute of Human Genetics^{27 (link)}.