DRIP_CTRL_24h_s
DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma; H7904) for 24h. Total nucleic acids were extracted with 0,5% %SDS /Proteinase K (Thermo Fisher Scientific, Waltham, MA) treatment at 37°C overnight and recovered by phenol-chloroform extraction and ethanol precipitation. DNA was digested by a restriction enzyme cocktail (20 units each of EcoRI, HindIII, BsrGI, XbaI) (New England Biolabs) in 1× NEBuffer 2, with or without RNase H treatment, overnight at 37°C. Fragmented DNA was cleaned by phenol-chloroform extraction and ethanol precipitation followed by two washes with 70% ethanol. Air-dried pellets were resuspended in 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE). 4 µg of digests was diluted in 450 µL of TE, and 10 µL was reserved as input for qPCR. 50 µL of 10× IP buffer was added (final buffer concentration of 10 mM sodium phosphate, 140 mM sodium chloride, 0.05% Triton X-100) and 10 µL of S9.6 antibody (1 mg/ml, kind gift from F. Chedin, UC Davis). Samples were incubated with the antibody at 4°C for 2 hours on a wheel. 50 µL of Protein A/G Agarose (Pierce), previously washed twice with 700 µL of 1× IP buffer for 5 minutes at room temperature, were added and samples were incubated for 2h at 4°C on a wheel. Each DRIP was then washed three times with 700 µL 1× IP buffer for 10 minutes at room temperature. After the final wash, beads were resuspended in 250 µL of 1× IP buffer and incubated with 60 units of Proteinase K for 45 minutes at 55°C. Digested DRIP samples were then cleaned with phenol-chloroform extraction and ethanol precipitation. Air-dried DRIP pellets were resuspended in 45 µL of 10 mM Tris-HCl pH 8. For DRIP-seq, samples from 3 DRIP experiments were pooled and sonicated to an average size of 300bp using a Bioruptor (Diagenode) for 20 cycles of 30 s On, 30 s Off, High setting.  Library preparation was performed at EMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs).