alias: E-MTAB-5817:H3K36me2_mOHT_s
title: Illumina HiSeq 2500 sequencing; ChIP-Seq mapping of histone modifications at multiple DNA Double Strand Breaks
design: ChIP-Seq mapping of histone modifications at multiple DNA Double Strand Breaks
library_construction_protocol: DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) 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 4OHT (Sigma; H7904) for 4h For ChIP:Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping. Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (10 to 200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1%  IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). Libraries for H3K4me2, H4K20me1, H3K36me3, H3K79me2, H2Bub, H3K56ac, H4K16ac, H3K9me2, H4S1P, H3K36me2 and γH2AX were prepared BGI (Beijin Genomics Institute, Hong Kong) using proprietary protocols. Libraries for FK2 samples were prepared at GATC biotech (Konstanz, Germany) using proprietary protocols.  Libraries for H2BK120ac, H2AZ, H1, MacroH2A, H4K20me3, 53BP1, H4K12ac, H2AZac and H3 were prepared atEMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs).  Libraries for BLESS were prepared using TruSeq Nano DNA LT Library Preparation Kit (Illumina).
identifiers.namespace: Universite de Toulouse ; UPS ; LBCMCP ; 118 route de Narbonne, 31062, Toulouse, France;CNRS ; LBCMCP ; F-31062 Toulouse, France
identifiers.id: E-MTAB-5817:H3K36me2_mOHT_s
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: dose
attributes.value: none
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: Anti-Histone H3K36me2 antibody (Abcam ab9049)