The cell cycle synchronization protocol was adapted from a previous publication [36 (link)]. In brief, PIGA intr5_1 gRNA positive H1-iCas9 ESCs were seeded at a density of 2 × 105 cells per well in a 12-well plate. To synchronize the cells at the G2/M phase, a 16-h treatment with 100 ng/ml nocodazole (Abcam, Cat# ab120630) was administered. Subsequently, the cells were washed twice with prewarmed 1 × PBS and then cultured in fresh E8 medium for 4 h or 12 h to release cells in the G1 or S phase, respectively. Alternatively, the synchronized cells were treated with 2 µg/ml doxycycline to induce Cas9 expression and genome editing, followed by 10 days’ culture for LD analysis.
Cell cycle analysis was performed using a standard protocol. Initially, the cell pellets were fixed by adding cold 70% ethanol dropwise while vortexing and then incubated overnight at − 20°C. Subsequently, the cells were washed twice and resuspended in FACS buffer containing 200 µg/ml rNase. After a 20-min incubation at room temperature, the cells were washed once and resuspended in FACS buffer containing 1 µg/ml propidium iodide (PI). Following a 10-min incubation at room temperature, the samples were ready for FACS analysis.
Cell cycle analysis was performed using a standard protocol. Initially, the cell pellets were fixed by adding cold 70% ethanol dropwise while vortexing and then incubated overnight at − 20°C. Subsequently, the cells were washed twice and resuspended in FACS buffer containing 200 µg/ml rNase. After a 20-min incubation at room temperature, the cells were washed once and resuspended in FACS buffer containing 1 µg/ml propidium iodide (PI). Following a 10-min incubation at room temperature, the samples were ready for FACS analysis.
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