Total nucleic acids were isolated from 106 cells by lysis in ice-cold buffer (20 mM Tris-HCl pH 7.5, 75 mM NaCl, 50 mM EDTA) and subsequent incubation with 200 µg/ml proteinase K (Roche) for 10 min on ice followed by addition of sarkosyl (Sigma) to a final concentration of 1%. Nucleic acids were sequentially extracted with TE-equilibrated phenol, phenol:chloroform:isoamyl alcohol (25:24:1), and chloroform, and then precipitated with isopropanol. Nucleic acids were collected by centrifugation, washed with 75% ethanol, air-dried and dissolved in nuclease-free water.
For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H231 (link) and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 0.01% BSA, 0.01% Triton X-100). Nucleic acids were ethanol-precipitated, dissolved in nuclease-free water and separated on a 0.7% agarose gel in 50 mM NaOH, 1 mM EDTA. After electrophoresis, the gel was neutralised in 0.7 M Tris-HCl pH 8.0, 1.5 M NaCl and stained with SYBR Gold (Invitrogen). Imaging was performed on a FLA-5100 imaging system (Fujifilm), and densitometry plots generated using an AIDA Image Analyzer (Raytest).
For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H231 (link) and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 0.01% BSA, 0.01% Triton X-100). Nucleic acids were ethanol-precipitated, dissolved in nuclease-free water and separated on a 0.7% agarose gel in 50 mM NaOH, 1 mM EDTA. After electrophoresis, the gel was neutralised in 0.7 M Tris-HCl pH 8.0, 1.5 M NaCl and stained with SYBR Gold (Invitrogen). Imaging was performed on a FLA-5100 imaging system (Fujifilm), and densitometry plots generated using an AIDA Image Analyzer (Raytest).
