DNA Double Strand Break Repair Assay

HeLa cells with pDRGFP (HR plasmid construct) and pimEJ5GFP (NHEJ plasmid construct) were transfected with pCBASceI, a I-SceI endonuclease expression vector with a mammalian promoter to introduce a DSB at a genomic I-SceI site of DNA repair plasmid constructs. Cells were grown for 4 days followed by flow cytometry analysis with Alexa Fluor 488 and mCherry channel. mCherry was used as a transfection control, pcDNA3-EGFP was an EGFP control, and HPRT was a negative control (26). Colony that posed superior amount of EGFP signal was chosen for next experiments with drug treatments. The transfections of pCBASceI, mCherry2-C1, and all other control plasmids were done. For the experiment with drug treatments, 300,000 HeLa cells per well were plated on six-well plates on day 1, followed by pCBASceI and control transfections on day 2 and drug treatment on day 3. Additionally, the repair assay was also analysed with live imaging with phase contrast and GFP channel (Supplementary Tables S4, S6).