DSB Reporter Assay

NHEJ and HR reporters were previously described.^31,32 Briefly, 10 μg of NHEJ reporter or HR reporter cassette were linearized by 50 U of NheI in a 50 μL reaction for 4 hours in 37°C water bath. Linearized DNA was gel purified and 1 μg of clean and linearized plasmid was transfected into U87 cells by using Lipofectamine3000 according to manufacturer’s instruction. Cells with chromosomally integrated reporter were selected by 1 mg/mL geneticin 3 days after transfection for 2 weeks. Stable transfected cells were seeded at 3x10⁵ cells/mL in a 6-well plate and 2µg/well of I-SceI coding plasmid was transfected into the cell by lipofectamine 3000 and incubated for 48h. Cells were harvest and GFP positive cells, which indicating successful NHEJ repair, were count by flow cytometry (BD FACSCelesta™ Flow Cytometer).