Cell Culture Protocols for Multiple Cell Lines

RPE1-hTERT, HCT116, and 293T cells were purchased from American Type Culture Collection. HeLa T-Rex Flp-In cells orginated from the laboratory of Stephen Taylor and were a kind gift from Laurence Pelletier. RPE1-hTERT-derived cell lines, 293T-derived cell lines, and HeLa T-Rex Flp-In-derived cell lines were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco/Thermo Fisher, 11965092) supplemented with 10% fetal bovine serum (FBS) (Wisent), 1× GlutaMAX, 1× nonessential amino acids (both Gibco/Thermo Fisher), and 100 U/mL penicillin and 100 µg/mL streptomycin (Pen/Strep) (Wisent). SUM149PT Cas9 cell origin and culture conditions were as described previously (Zimmermann et al. 2018 (link)). HCT116-derived cell lines were grown in McCoy's 5A (Gibco, 1660-082) + 10% FBS (Gibco) + 1% penicilllin–streptomycin mix (Nakalai Tesque, 09367-34) + 1× L-glutamine (final 2 mM; Wako Chemical, 073-05391). RPE1-hTERT Cas9 TP53-KO BRCA1-KO were cultured at 37°C and 3% O₂. All other cell lines were grown at 37°C and atmospheric O₂. All cell lines were routinely authenticated by STR and tested negative for mycoplasma.