NextSeq 500 sequencing; DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
gammaH2AX_pOHTpATMi_s
For ChIP: Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping. 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. For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h. For ATM inhibition, cells were pretreated for 1 h with 20μM KU-55933 (Sigma, SML1109) and during subsequent 4OHT treatment. For ATR inhibition, cells were pretreated for 1 h with 2μM ETP-46464 (Sigma, SML1321) and during subsequent 4OHTsiRNA transfection were performed using the 4D Nucleofector X (kit SE cell line) according to the manufacturer's instructions and subsequent treatment(s) were performed 48 h later. siRNA sequences: CTRL: CAUGUCAUGUGUCACAUCU    SCC1: GGUGAAAAUGGCAUUACGG   WAPL: CGGACUACCCUUAGCACAA. For CRISPR/Cas9-mediated DSB induction, sgRNA (AsiSI site position: “CGCCGCGATCGCGGAATGGA” or Position further within the TAD=\"CRISPRsite\": “GGGCCAGTCGCGGCACTCGC”) were delivered in U2OS cells using the “nanoblades” technology which relies on direct cell transduction with a viral-derived particle containing the Cas9/sgRNA ribonucleoprotein (Mangeot et al, 2019). Cells were analyzed 24 h after transduction.    For ChIP: Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). For the calibrated ChIP-seq of SCC1, we used a spike-in method as in Kojic et al (2018). Briefly, cross-linked cells were first lysed 10min at 4°C in 500 µL of the Lysis Buffer 1 (10mM Tris pH8, 10mM NaCl, 0.5% NP-40, Complete protease inhibitor (Sigma)) then 10min at 4°C in the Lysis buffer 2 (50mM Tris pH8, 10mM EDTA, 0.5% NP-40, Complete protease inhibitor (Sigma)) and subsequently sonicated in 15ml conical tubes with a Bioruptor Pico (Diagenode) in the presence of 800 mg of sonication beads (20 cycles of 30 sec ON/30 sec OFF) to an average fragment size of 250pb. Prior to immunoprecipitation with SCC1 antibody, 20% of chromatin from mouse ES cells (40µg) was added to chromatin prepared from treated or untreated human DIvA cells (200µg).  Antibodies : pATM (S1981): Abcam ab81292 (2µg) SCC1: Abcam ab992 (4µg) MDC1: Abcam ab11171 (3µg) For ChIP: Sequencing libraries were prepared from IP of SCC1 and pATM(S1981) and input samples using the NEBNext® Ultra™ II Library Prep Kit (NEB)for Illumina. MDC1 libraries were prepared at EMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs).