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
ChIP-seq: Briefly, cells were crosslinked with 1% formaldehyde for 15 min at room temperature and quenched with 0,125 M glycine for 5 min. Cells were washed with 1X PBS and harvested by scrapping. Cell pellets were resuspended in cellular lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% NP-40) and homogenized with a Dounce homogenizer. Nuclei were harvested by centrifugation and resuspended in nuclear lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) 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 ChIP-seq: Nuclei were sonicated using a Branson Sonifier 250 (at a power setting of 5 and 50% duty cycle) to obtain fragments of about 500 bp. Samples were diluted 10 times in dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) and each aliquot of 200 µg of chromatin was precleared with 100 µL of 50% protein-A (Pierce) and protein-G (Sigma) beads, (previously blocked with 500 µg of BSA) during 2 hours at 4°C on a rotating wheel. 100 µL of pre-cleared chromatin was kept at -20°C as input sample and 10-200 µg was incubated with specific antibodies at 4°C on a rotating wheel overnight (antibodies and corresponding amount of chromatin are detailed in Supplementary Table 2). The next day, 100 µL of pre-blocked protein A/G beads were added to each sample for 2 hours at 4°C on a rotating wheel. Beads were washed once with dialysis buffer (50 mM Tris pH 8.1, 2 mM EDTA, 0.2% Sarkosyl), five times with wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% NP-40, 1% NaDoc) and twice in TE buffer (Tris 10 mM pH8, 1 mM EDTA). Immunoprecipitated complexes were resuspended in 200 µL of TE and input samples were adjusted to 200 µL. Next, 0.5 µL of RNase A (30mg/mL) was added during 30 min at 37°C. Following addition of 0.5% SDS, samples were incubated overnight with shaking at 70°C to reverse the crosslink. Next, proteinase K was added (200 µg/mL final) and samples were incubated for 1h30 at 45°C. DNA was purified by phenol:chlorophorm extraction followed by ethanol precipitation. Samples were resuspended in 100 µl water. ChIP-seq: multiple ChIP experiments were pooled, sonicated for 10-15 cycles (30 sec on, 30 sec off, high setting) with a Bioruptor (Diagenode), then concentrated with a vacuum concentrator (Eppendorf). For SPIN1, H3K4me3, total RNAPII, RNAPII S2P, RNAPII S5P, RNAPII S7P, RNAPII Y1P, POLR3A and POLR3E ChIP-seq, about 10 ng of purified DNA was subjected to library preparation (NEBNext Ultra II DNA Library kit, New England Biolabs)