Whole genome sequencing of RNaseH2 deficient cells RPE1 cells and mouse tumours.

To study the effect of defective ribonucleotide excision repair in mammals on mutational processes we performed whole genome sequencing (WGS) of human RPE1 cells and mouse tumours deficient in RNaseH2. WGS of TP53-KO hTERT-RPE1 cells with and without loss-of-function mutations in RNASEH2A or RNASEH2B, introduced by CRISPR/Cas9 was performed before and after bottlenecking. Ancestral populations for RNase H2 wild-type and null cells were established after initial single cell sorting, and clones grown for approximately 100 generations. Single cell sorting was performed every 25 generations, creating bottlenecks to capture accumulating mutations. Ancestral and endpoint cultures were sequenced to determine mutations that had accumulated during this experiment using Illumina TruSeq PCRFree High Throughput sequencing. RNaseH2 deficient mouse tumours and matched RNaseH2 proficient liver samples were taken from Villin-Cre+ Trp53fl/fl Rnaseh2bfl/fl mice with epithelial-specific deletion of Trp53 and Rnaseh2b on a C57Bl/6J background. DNA from tumour and liver samples were sequenced by WGS at ~60X and ~30X respectively using Illumina DNA PCR-Free Library Prep.