Tumor RNA
RNA was extracted from 1mm punch biopsy cores of formalin FFPE tumor blocks using CELLDATA DNAstorm/RNAstorm 2.0 Combination Kit (Biotium) according to the manufacturer's instructions with minor modification. The resulting eluate was incubated with 28 U DNase I (RNase-Free DNase Set, Qiagen) for 30 minutes at room temperature to digest DNA. The digested eluate was purified using the Zymo RNA Clean & Concentrator kit and stored at -80°C. RNA concentration was quantified using Nanodrop and 100 ng of RNA was used for library preparation. For samples with less than 100ng of RNA, all extracted tumor RNA was used for library preparation (range 25-100 ng). Double-stranded complementary DNA (cDNA) was synthesized from tumor RNA using the NEBNext Ultra™ II RNA First-Strand Synthesis Module and Non-Directional Second Strand Synthesis Module (New England Biolabs). Double-stranded cDNA was treated with 100 U S1 nuclease (Thermo Fisher) for 30 minutes at room temperature. The KAPA Hyper Prep kit (Kapa Biosystems) was used to prepare libraries for sequencing, following manufacturer's instructions with slight modifications as previously described.62 Whole coding transcriptome capture was performed using the Twist Biosciences Comprehensive Exome Hybridization kit, following the manufacturer's instructions. Captured libraries were sequenced using 2x150-bp paired-end reads on Illumina HiSeq4000 or NovaSeq6000.