title: RNA-Seq of human
design: The human metabolic enzyme sgRNA library targets 1773 metabolic enzyme genes (4 sgRNAs per gene). The sgRNAs were designed and synthesized by GENEWIZ (Suzhou, China) and then packaged into the lentiCRISPR transfer plasmid as previously described in the Zhang laboratory (46). For virus production, 293T cells were cotransfected with 5 g of lentiCRISPR plasmid library, 4 g of psPAX2 and 3 g of pVSVg (Addgene, Watertown, MA, USA) with 30 l of Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific). The transfected 293T cells were incubated at 37C, and the transfection medium was replaced after 8 h. Viral particles were harvested 48-72 h after transfection and frozen at -80C until further use. For the in vivo CRISPR-Cas9 knockout screen under oxaliplatin treatment, 4107 DLD1 cells were plated onto fifteen 10 cm dishes to ensure sufficient coverage of sgRNAs and infected at a low multiplicity of infection (MOI 0.3) with 10 g/mL polybrene to ensure that most cells received only one viral construct with high probability. At 48 h after infection, the infected DLD1 cells were selected with 2 g/ml puromycin for 7 days to select the positively transduced cells and eliminate uninfected cells in order to obtain genome-edited cell pools. The selected cells were subcutaneously injected into the flanks of female BALB/c nude mice (4 to 5 weeks old, Beijing Vital River Laboratory Animal Technology). When tumors became palpable, the tumor-bearing mice were divided randomly into two groups (6 mice per group), and treated with PBS or oxaliplatin (5mg/kg) by intraperitoneal (i.p.) injection every 3 days. On day 30, we excised the tumors and weighted them before performing genomic DNA extraction using a DNeasy Blood & Tissue Kit (69504, Qiagen, Dusseldorf, Germany). DNA fragments containing the sgRNA sequences were amplified by 2-step PCR using NEBNext Ultra II Q5 Master Mix (M0544L, New England Biolabs, Ipswich, MA, USA) and the primers listed in Supplementary Table S4. The PCR products containing the sgRNA sequences were gel extracted, quantified, mixed, sequenced and analyzed as previously described(46).