RNase HII-Aided Comet Assay for DNA Breaks

Neutral comet assays were performed according to manufacturer’s protocol (Trevigen) with the addition of an RNase HII digestion step. Cells were harvested 72 hr after Hit & Run pMMP Cre retroviral infection^{65 (link)}. Following cell lysis, slides were flooded with 1 X ThermoPol Buffer (NEB) for 5 minutes at 25 °C to equilibrate and digested with 10U RNase HII for 1 hr at 37 °C. To measure DNA breaks following RNase HII digestion, slides were stained with SYBR Green (1:10,000) in PBS. Comet tail moments were visualized or Axio Observer.A1 fluorescence microscope (Carl Zeiss), equipped with a Plan- Apochromat 40× NA-1.4 oil objective, the AxioCam CCD camera, and the AxioVision Rel Version 4.7 software and scored with OpenComet software^{78 (link)}.