NextSeq 500 paired end sequencing; ATM-dependent formation of a novel chromatin compartment regulates the Response to DNA Double Strand Breaks and the biogenesis of translocations
ATM-dependent formation of a novel chromatin compartment regulates the Response to DNA Double Strand Breaks and the biogenesis of translocations
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 or 24h. For ATM inhibition, cells were pretreated for 1 h with 20μM KU-55933 (Sigma, SML1109) and during subsequent 4OHT treatment. For DNA-PK inhibition, cells were pretreated for 1 h with 2μM NU-7441 (Selleckchem, S2638) and during subsequent 4OHT. siRNA 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; or SCC1: GGUGAAAAUGGCAUUACGG:   or SMC1 (siSMC1): UAGGCUUCCUGGAGGUCACAUUUAA; or 53BP1 (si53BP1): GAACGAGGAGACGGUAAUA; or SUN2 (siSUN2): CGAGCCTATTCAGACGTTTCA; or ARP2 (siARP2): GGCACCGGGUUUGUGAAGU or SETX (siSETX): GAGAGAAUUAUUGCGUACU. For cell synchronization, cells were incubated for 18 h with 2 mM thymidine (Sigma, T1895), then released during 11 h, followed by a second thymidine treatment for 18 hr. S, G2 and G1 cells were then respectively treated with OHT at, 0, 6 or 11 h following thymidine release and harvested 4 h later. 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.