Transcriptional repression accounts for RNA:DNA hybrids accumulation at DNA Double Strand breaks
Transcriptional repression accounts for RNA:DNA hybrids accumulation at DNA Double Strand breaks
qDRIP-seq was adapted from Crossley et al, NAR, 2020. In brief, 2.5 × 10 6 of trypsinized DIvA cells were mixed to 1.67 × 10 6 Drosophila S2 cells DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. AsiSI dependent-DSB were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h.siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection qDRIP-seq was adapted from Crossley et al, NAR, 2020. In brief, 2.5 × 10 6 of trypsinized DIvA cells were mixed to 1.67 × 10 6 Drosophila S2 cells and lysed overnight at 37°C in TE buffer containing 0.5% SDS and 800 μg proteinase K (Roche, 03115828001). DNA was extracted by phenol:chloroform extraction using phase lock tubes (Qiagen, MaxTract, 129065) followed by ethanol precipitation. DNA was resuspended on ice in 130 μl TE buffer before sonication in Covaris S220 (microtubes PN520045) to obtain ~300 bp DNA fragments (Covaris S220, 140W peak incident power, 10% Duty factor, 200 cycles per burst for 80 sec). Immunoprecipitation was performed in triplicate by incubating 4 μg of sonicated DNA with 10 μg of S9.6 antibody (Antibodies Incorporation) in 1x binding buffer (10 mM NaPO 4 , 140 mM NaCl, 0.05 % Triton X100) overnight and at 4°C. Agarose A/G beads (Thermo Scientific, 20421) we added to samples for 2 h at 4°C. Beads were washed 4 times in binding buffer followed by incubation with elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, 0.3 μg/μL proteinase K) for 45 min at 55°C. Samples were subjected to phenol:chloroform extraction and ethanol precipitation, and were resuspended in low EDTA TE. Sequencing libraries were prepared using the Swift ACELL-NGS 1S Plus kit according to the manufacturer's instructions using 12 PCR cycles. Libraries were pooled at equimolar concentrations.