Quantification of Homologous Recombination Efficiency

A Schematic diagram of HR repair assay is shown in Supplementary Fig. 1a. DR-GFP, pCAGGS, and pCBASce plasmids were kindly provided by Dr. Maria Jasin (Memorial Sloan-Kettering Cancer Center, New York, NY). U2OS cells containing a single copy of the HR repair reporter substrate DR-GFP in a random locus were generated as previously described^{12 (link)}. GFP-expressing plasmid (pEGFP-C1) was used for transfection efficiency control. Twenty-four hours after ZNF668 siRNA, TTK plasmid, or BRCA1 siRNA transfection, cells were re-seeded; the next day, cells were transfected with pCBASce plasmids. For cell lines that do not stably contain the DR-GFP plasmid, 1×10⁶ cells were electroporated with 12 μg of DR-GFP and 12 μg of pCBASce plasmids at 270V, 975uF using a BioRad genepulsar II^{45 (link)}. 48 to 72 hours later, flow cytometry analysis was performed to detect GFP-positive cells using a FACScalibur apparatus with CellQuest software (Becton Dickinson, San Jose, CA). Unless otherwise specified, results were mean + SD from three independent experiments.