Following FACS or puromycin selection, single cells were permitted to expand for 7–14 days to establish clonal populations. Colonies were manually picked and replated into individual wells of a 96-well plate. Once the wells reached 80–90% confluence, cells were dissociated with Accutase and split at a 1:3 ratio to create a frozen stock and two working stocks that were maintained in culture. For genomic DNA isolation, cells from one of the working stocks were lysed in 50 μL lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% Sarcosyl) with 40 μg/mL Proteinase K (New England BioLabs) for 1–2 hours in a humidified incubator at 56°C. Genomic DNA was precipitated by addition of 100 μL 95% ethanol with 75 mM NaCl, followed by incubation at −20°C for 2 hours. Precipitated DNA was washed three times with 70% ethanol, resuspended in 30–50 μL TE buffer with 0.1 mg/mL RNase A (Thermo Fisher Scientific), and allowed to dissolve at room temperature overnight. hPSC clones were screened by PCR amplification of a small region surrounding the targeted site using
