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. Hi-C experiments were performed in DIvA cells using the Arima Hi-C kit (Arima Genomics) according to the manufacturer's instructions. 1 million cells were used by condition. Briefly, cells were cross-linked with 2% formaldehyde for 10 min at RT, lysed and chromatin was digested with two different restriction enzymes included in the kit. Ends were filled-in in the presence of biotinylated nucleotides, followed by subsequent ligation. Ligated DNA was sonicated using the Covaris S220 to an average fragment size of 350 bp with the following parameters (Peak incident power: 140; Duty factor: 10%; Cycles per burst: 200; Treatment time: 70s). DNA was then subjected to a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter). Biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit). Hi-C library was prepared on beads using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina (New England Biolabs) following instructions from the Arima Hi-C kit.