Generation of Cas9 stable cells

lentiCas9-Blast viral particles were purchased from Addgene (#52962) and used to infect U2OS and HeLa cells according to the manufacturer’s protocol. Briefly, on the day of infection, cells were trypsinized, counted, and diluted to a working concentration of 50,000 cells mL⁻¹ in DMEM media supplemented with 10% FBS/1× pen-strep/1× glutamine (Gibco) (DMEM complete) and 10 µg mL⁻¹ polybrene. Viral particles were serially diluted down to 1:500 from the original stock (2.5 × 10⁵ TU mL⁻¹), with 500 µL of each dilution added to the corresponding wells of a 6-well plate. 1 mL of cell suspension was added to each well and incubated at 37 °C and 5% CO₂. 48 h after infection, selection was performed in DMEM-complete media supplemented with 10 µg mL⁻¹ Blasticidin S HCl (Gibco). After selection, cells stably expressing Cas9 were maintained in DMEM-complete media with 5 µg mL⁻¹ Blasticidin S HCl.