Characterization of U2OS DNA Repair Cells

U2OS cells (ATCC), U2OS DSB repair pathway reporter cells [DR-green fluorescence protein (GFP), SA-GFP, EJ5-GFP, and EJ2-GFP were a gift from Dr. Jeremy Stark, City of Hope, Duarte, California, USA] were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS; Sigma) and antibiotics (10 U/mL penicillin and 10 μg/mL streptomycin; ThermoFisher) at 37 °C and 5% CO₂. Stable U2OS shRNA knockdown cells were generated by transfection with HuSH 29-mer shRNA expression vectors (Origene) using GenJet U2OS Transfection Reagent (SignaGen) followed by selection with puromycin (InvivoGen) 48 h post-transfection until colonies formed. Colonies were then isolated, expanded, and screened for knockdown by Western blot and RT-qPCR. Knockdown efficiency of siRNA and shRNA targeting sequences have been previously characterized and their sequences made available (Dilworth et al. 2018 (link)).