NextSeq 500 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. 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. 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.  The 4C-seq experiments were realized as in Matelot and Noordermeer (2016) with minor modifications. Briefly, 15 million DIvA cells were cross-linked with 2% formaldehyde for 10 min at RT and the reaction was stopped using glycine (0,125M final) for 5min on ice. Cells were lysed, permeabilized with A50µL SDS 0,5% and digested with MboI (New England Biolabs). Three rounds of 4 h of digestion with MboI were necessary. Digested DNA was then ligated with a T4 DNA ligase (HC) (Promega), purified and digested with NlaIII overnight (New England Biolabs). After a second ligation step, DNA was purified before proceeding to the library preparation. For DNA purification steps, AMPure XP beads (Beckman Coulter) were used diluted at 1:10 in 20% PEG solution (PEG 8000 (Sigma) 20%, 2.5M NaCl, Tween 20 20%, Tris pH8 10mM, EDTA 1mM).  For 4C-seq library preparation, 800ng-900ng of 4C-seq template was amplified using 16 individual PCR reactions with inverse primers (PAGE-purified) including the Illumina adapter sequences and a unique index for each condition. Libraries were purified with the QIAquick PCR Purification Kit (Qiagen). The primer sequences used to amplify each viewpoint were:  Ctrl: FW:ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCAGGTTATCATCCCAA REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACCTTCGCTGTACCTTTG DSB1: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCTGGCAACTTATGAATCAGGA REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGTCAAAAGCCAAGGGGACA DSB2: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTTACGATTATTTGTGAATTTTG REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGCTAATTCTGAGTTACATACATT DSB3: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGATTACGTAGAAGGGTGCC REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGGCAAATGATAACCCTGT DSB4: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTTATACTAAGATGTCAGTTCCT REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACGCACCTGGTTTAGATT