Illumina HiSeq 2500 paired end sequencing; ChIP-Seq mapping of histone modifications at multiple DNA Double Strand Breaks
ChIP-Seq mapping of histone modifications at multiple DNA Double Strand Breaks
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 BLESS: After 4OHT and or auxin treatment in G1 synchronized cells, DSBs labelling was carried out using BLESS method as described previously (Crosetto et al, Nath Methods, 2013). Briefly, cells were fixed with formaldehyde, lysed and mildly digested with proteinase K at 37C in order to purify intact nuclei, then DSBs were blunted and 5'phosphorylated using Quick Blunting Kit (NEB). Subsequently, a biotinylated proximal linker (Sigma) was ligated to DSBs using T4 ligase (NEB). Next, DNA was extracted by precipitation with isopropanol and sonicated using Covaris S220 ultrasonicator to create fragments approximately 400bp long. Labeled, biotinylated fragments were captured on streptavidin beads (Invitrogen) and ligated to a distal linker (Sigma). Resulting DNA fragments were then linearized by I-SceI (NEB) digestion and amplified by PCR. 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).