NHEJ Repair of I-SceI-induced DSBs

HEK293 cell lines containing a stably integrated copy of the EJ5-GFP reporter were used to measure the repair of I-SceI-induced DSBs by NHEJ (Bennardo et al. 2008 (link)). Briefly, 48 h after siRNA transfection, cells were cotransfected with a mCherry expression vector and the I-SceI expression vector pCBASce. Forty-eight hours later, the percentage of GFP-positive cells among mCherry-positive cells was determined by FACS on a BD LSRII flow cytometer (BD Bioscience) using FACSDiva software version 5.0.3. Quantifications were performed using WinMDI 2.9 (freeware), FACSDiva (BD Biosciences), or FlowJo software (Flowing Software 5.2.1.).