For the HU-induced HR assay, reporter cells underwent initial double thymidine block (2 mM, two cycles of 16-hour in drug with a 12-hour interval of drug-free medium in between) to enrich the population in S-phase, followed by treatment with 2 mM HU for 24 h. For the I-SceI-induced HR assay, reporter cell lines were infected with lentiviruses encoding HA-I-SceI. Four days after HU or I-SceI expression, EGFP-positive events were quantified by FACS analysis using a BD Accuri C6 flow cytometer and accompanying data analysis software (CFlow, Becton-Dickinson).
Analysis of HR used in the HR-Flex/D-Flex reporter and HR-Luc/D-Luc reporter was performed as previously described (11 (link),31 (link)). Briefly, genomic DNA was extracted four days after I-SceI viral infection, followed by PCR amplification. The PCR products were digested with or without BamHI and EcoRI and resolved by 1.5% agarose gel electrophoresis. The percentage of restriction enzyme digestible PCR products relative to the total DNA provided an estimation of HR efficiency, determined by quantifying the DNA bands using ImageJ.
Analysis of HR used in the HR-Flex/D-Flex reporter and HR-Luc/D-Luc reporter was performed as previously described (11 (link),31 (link)). Briefly, genomic DNA was extracted four days after I-SceI viral infection, followed by PCR amplification. The PCR products were digested with or without BamHI and EcoRI and resolved by 1.5% agarose gel electrophoresis. The percentage of restriction enzyme digestible PCR products relative to the total DNA provided an estimation of HR efficiency, determined by quantifying the DNA bands using ImageJ.
