MSK-IMPACT targeted DNA-Seq of DUC18828
Patient tumor was sequenced using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform.21 This same platform was used to analyze the DUC18828 cell line. Genomic DNA was extracted using the Qiagen DNA extraction kit (Cat# 13343). DNA was then sent to MSKCC, de-identified, for analysis.   ddPCR was used to determine the percentage of human cells in the PDX sample. Specifically, 9ng of gDNA from the sample was combined with primers specific to Mus musculus and Homo sapiens PTGER2, FAM- and HEX-labeled probes, MseI, and digital PCR Supermix for probes (no dUTP). The reaction was performed on a QX200 ddPCR system (Bio-Rad catalog # 1864001) and partitioned into ~16,000 droplets using the QX200 droplet generator. The emulsified PCR was run on a 96-well thermal cycler (95C 10; 40 cycles of 94C 30 and 60C 1; 98C 10; 4C hold). The plate was read and analyzed with the QuantaSoft software to assess the number of droplets positive for human or mouse. Human percentage was calculated at 99.6%.  After PicoGreen quantification, 100ng of DNA were used to prepare libraries using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with 8 cycles of PCR. 110-160ng of each barcoded library were captured by hybridization in a pool of 6 samples using the IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) assay (Nimblegen SeqCap), designed to capture all protein-coding exons and select introns of 505 commonly implicated oncogenes, tumor suppressor genes, and members of pathways deemed actionable by targeted therapies. Captured pools were sequenced on a HiSeq 4000 in a PE100 run using the HiSeq 3000/4000 SBS Kit (Illumina) producing an average of 502X coverage per sample.