Alkaline Gel Electrophoresis of RNase-Treated DNA

Genomic DNA was treated with RNase H2 and separated by alkaline gel electrophoresis, essentially as described (Benitez‐Guijarro et al., 2018 (link)). In brief, genomic DNA (250 ng) was treated with 1 pmol of purified recombinant human RNase H2 (Reijns et al., 2011 (link)) and 0.25 μg of DNase‐free RNase (Roche) for 1 hr at 37℃ in 100 μl reaction buffer (60 mM KCl, 50 mM Tris–HCl pH 8.0, 10 mM MgCl₂, 0.01%Triton X‐100). Nucleic acids were ethanol precipitated and separated by alkaline gel electrophoresis (0.7% agarose, 50 mM NaOH, 1 mM EDTA). After electrophoresis, the gel was neutralized in 0.7 M Tris–HCl pH 8.0, 1.5 M NaCl and stained with SYBR Gold (Invitrogen). Images were taken using the FLA‐5100 imaging system (Fujifilm), and densitometry plots generated using AIDA Image Analyzer (Raytest).