Illumina HiSeq 1000 paired end sequencing; Principle of DNA Double Strand Breaks clustering revealed by Capture-HiC
Principle of DNA Double Strand Breaks clustering revealed by Capture-HiC
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 AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. Capture Hi-C library generation was carried out as described previously 2. Briefly, 30.106 cells were fixed in 2% formaldehyde for 10 minutes. After overnight digestion with HindIII, DNA ends were labeled with biotin-14–dATP using the klenow fragment of DNA pol I and religated in preserved nuclei. 50 ug of DNA was sheared to an average size of 450 bp (Covaris). Sheared DNA was then end repaired, adenine tailed and double size selected from 250 to 600 bp (AMPure XP beads). Chimerae marked by biotin were pulled down using MyOne Streptavidin C1 DynaBeads (Invitrogen) and ligated to PE adaptors (Illumina). The Hi-C libraries (retained on the beads) were amplified using PE primers (Illumina) with 10 PCR amplification cycles. Capture was next carried out on Hi-C library, with SureSelect target enrichment, using the custom-designed biotinylated RNA bait library and custom paired-end blockers according to the manufacturer's instructions (Agilent Technologies). After library enrichment by capture, a post-capture PCR amplification step was carried out using PE PCR 1.0 and PE PCR 2.0 primers with 7 PCR amplification cycles. Captured-Hi-C libraries were performed on damaged (4OHT treated) or undamaged (untreated) samples in biological duplicates (BR#1 and BR#2) and were sequenced on the Illumina HiSeq 1000 platform.
Experimental Factor: undamaged DNA
genetic modification