Amplicon-seq_D_siCtrl_mOHT_p
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.  Cells were then lysed in cytoplasmic lysis buffer (50mM HEPES pH7.9, 10mM KCl2, 1.5mM MgCl2, 0.34M sucrose, 0.5% triton X-100, 10% glycerol, 1mM DTT) for 10 minutes on ice, then washed once in cytoplasmic lysis buffer before lysis in genomic extraction buffer (50mM Tris pH8.0, 5mM EDTA, 1% SDS, 0.5mg/mL proteinase K). Lysate was incubated at 60°C for 1 h. Genomic DNA was then ethanol precipitated on ice for 1h, pelleted at 19,000g for 20 min and washed twice in 75% ethanol. Genomic DNA was then used in a multiplex PCR reaction that amplified 25 target sites; 20 AsiSI cut sites and 5 uncut control sites.  Amplicons were size selected using SPRIselect beads (Beckman, B23318) and subjected to DNA library preparation via the NEBNext Ultra II kit (NEB, E7645L).