Exome and RNA Sequencing
tumor
TUMOR
DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) tumor biopsies using the Covaris truXTRAC FFPE total NA Plus (Column) kit and the Covaris M220 focused ultrasonicator (Covaris), respectively, according to the manufacturer's protocol with several modifications. Two 15 μm scrolls were sectioned from FFPE blocks by the UCLA Translational Pathology Core Laboratory, and each scroll was individually processed for paraffin emulsification, proteinase K, and centrifugation. RNA-containing supernatants or DNA-containing tissue pellets from two individual scrolls were combined in their respective columns for washing and elution. RNA quality and quantity were evaluated using the Agilent Bioanalyzer RNA Pico kit (Agilent Technologies), and DNA quality and quantity were assessed using the Agilent Tapestation 4200 and Genomic DNA screen tape (Agilent Technologies).RNA (RNA-seq) and whole-exome sequencing (WES) library preparation were automated on the Agilent BRAVO Platform (Agilent Technologies). WES libraries were prepared using the Agilent SureSelect XT HS2 DNA library preparation system, with hybridization capture by the Agilent SureSelect v7 Exome with the Agilent OneSeq CNV Backbone panel according to the manufacturer's protocol. RNA-seq libraries were prepared using the Agilent SureSelect XT HS2 RNA library preparation system, with hybridized capture of cDNA using the Agilent SureSelect v7 Exome reagent. Pools (2 nmol/L) of either WES or RNA-seq libraries were sequenced by the UCLA Technology Center for Genomics and Bioinformatics core laboratory on the Illumina Novaseq (S4 flow cell, 2 × 150 bp reads).