title: ENDseq2_siCTRL_mOHT
organism: Homo sapiens
description: Protocols: ENDseq: Briefly, 5-7x106 DIvA cells per condition were collected by trypsination. 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. Total RNA spike-ins were prepared from mid-log phase S. cerevisiae (BY4741 strain) cells grown in YPD + 2% glucose and labeled with 5 mM 4-thiouracil (4TU, Sigma-Aldrich, 440736) for 5 min. AsiSI dependent-DSB were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h. Etoposide (Sigma, E1383) was used at 50 µM for 30 min for END-seq, 50 µM for 1 h for ChIP. siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection ENDseq:cells were embedded into two or three 0.75% agarose plugs (BioRad CHEF Mammalian genomic DNA Plug Kit). Plugs were subjected to proteinase K and RNAse A treatments. Exonuclease VII and T digestion (NEB) were carried out followed by the addition of Klenow fragment (3'-5' exo-) for 1 h at 37°C. Next, ligation of DNA ends with 10 μM of pre-annealed END-seq adapter 1 (see107 for sequence) was achieved. Plugs were melted and dialysis performed. DNA was next subjected to a quick proteinase K digestion before being transferred into Covaris S220 microtubes (PN520045) for DNA fragmentation (Covaris S220, 175W peak incident power, 10% Duty factor, 200 cycles per burst for 240 sec). Fragmented DNA was precipitated using ethanol and glycogen. Next, the fragmented DNA was added to Dynabeads MyOne magentic C1 beads (ThermoFisher) for 30 min at room temperature. After washing, samples were incubated with an end repair mixture of the large Klenow fragement, T4 PolI and T4 PNK. Subsequent washing and Klenow fragment (3'-5' exo-) reaction was performed. This was followed by ligation with of END-seq adapter 2 and the addition of the USER enzyme (NEB).  ENDseq:20 cycles of PCR reaction were achieved with NEBNext multiplex oligos for Illumina (E7335S, New England Biolabs) and PCR products were purified with SPRIselect or Ampure XP beads (Beckman Coulter) using first a size selection with a 0.6X beads-to-sample ratio followed by two consecutive purifications with a 0.9X ratio.