Illumina HiSeq 1000 paired end sequencing; Genome wide mapping of long range contacts unveils DNA Double Strand Breaks clustering at damaged active genes.
Genome wide mapping of long range contacts unveils DNA Double Strand Breaks clustering at damaged active genes.
DIvA (AsiSI-ER-U20S)cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) and 1 μg/mL puromycin (DIvA cells) at 37 °C under a humidified atmosphere with 5% CO2. For cell synchronization in G1, cells were incubated with 2 mM thymidine for 16 h, released for 14 h and subjected to the second thymidine treatment for 19 h. G1 cells (12h after release) were treated with 4OHT (4h) followed by an auxin treatment of 2h when indicated.  For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. After 4OHT and or auxin treatment in G1 synchronized cells, DSBs labelling was carried out using BLESS method as described previously (Crosetto et al, 2013). Briefly, cells were fixed with formaldehyde, lysed and mildly digested with proteinase K at 37ºC 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 were prepared using TruSeq DNA LT Sample Prep Kit (Illumina), followed by 2x60 bp next generation sequencing on Illumina HiSeq 2500.