Access array barcode library for sequencers

Fecal samples were collected from all the mice (N= 15/group) and frozen at −80°C until further use. A PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc.) was used for fecal DNA extraction, according to the manufacturer’s protocol [11 ]. First stage amplifications were performed followed by the second-stage PCR amplification with Access Array Barcode Library for Illumina Sequencers (Fluidigm, South San Francisco, CA; Item# 100–4876) at the University of Illinois at Chicago Sequencing Core. Sequencing was performed at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign. The gene amplicon sequence data generated as part of this study have been submitted to the NCBI BioProject database (PRJNA540508).

Single murine leukemia cells from mouse 322 that harboured frameshift mutations in both Sh2b3 and Nf1 were analysed to determine whether these mutations occurred in the same cells. Live, GFP⁺ cells from frozen murine bone marrow were single cell sorted into 96 well-plates on the BD Fusion. Control mouse cells that were not genome-edited were single cell sorted as a control for the single cell sequencing. Primers are listed in Table S5.
Single human or mouse cells were lysed and whole genome amplified using a REPLI-g Single Cell Kit (Qiagen). Primers for targeted re-sequencing of patient mutations were designed to include patient-specific heterozygous SNPs within the same amplicon (listed in Table S5). For the murine leukemia the primers from the CRISPR-Cas9 screening were used (listed in Table S5). Targeted amplification was performed using KAPA2G Fast Multiplex PCR Kit (Sigma-Aldrich), followed by addition of sample barcodes and sequencing adapters using FastStart High Fidelity PCR System (Roche) and Access Array™ Barcode Library for Illumina Sequencers (Fluidigm), as previously described (Quek et al., 2018 (link)). A total of 240 human patient and control cells (x3 amplicons) and 96 mouse leukemia and control cells (x2 amplicons) were sequenced on an Illumina MiSeq using a Nano Reagent Kit (Illumina) and 300-cycle paired-end sequencing.