Genome-wide DNA:RNA hybrid detection

DRIP was performed as previously described.⁵⁶ (link) Briefly, cells (1 × 107) were washed with ice-cold PBS and resuspended in 1.6 mL of TE buffer. Then, 50 μL of 20% (w/v) SDS and 5 μL of 20 mg/mL proteinase K were added and further incubated at 37 °C for 12 h. DNA lysate was poured directly into the Maxtract phase-lock gel tube (Qiagen) and 1 vol (1.6 mL) of phenol/chloroform/isoamyl alcohol (25:24:1) was added. DNA was precipitated with NaAc. The DNA pellet was air-dried and sobulized in TE buffer. Extracted genomic DNA was digested using a cocktail of restriction enzymes (BsrGI, EcoRI, HindIII, SspI and XbaI) at 37 °C overnight. Digested DNA was then isolated using phenol-chloroform precipitation. 8 μg of extracted DNA were diluted in DRIP binding buffer (10 mM sodium phosphate, pH 7, 140 mM NaCl and 0.05% (v/v) Triton X-100). Twenty micrograms of the S9.6 antibody were added to the diluted DNA and further incubated for 14–17 h at 4 °C. Then protein G PLUS-agarose (Santa Cruz Biotechnology Inc.) was added and incubated for 2 h at 4 °C. Precipitates were isolated using NucleoSpin Extract II (Macherey-Nagel).