title: Illumina HiSeq 2500 paired end sequencing; Hi-C and capture Hi-C for HPV16 infected human W12 cervical cell lines and normal cervical tissue
design: Hi-C and capture Hi-C for HPV16 infected human W12 cervical cell lines and normal cervical tissue
library_construction_protocol: W12 cells were cultured in a monolayer to restrict cell differentiation and maintain the phenotype of the basal epithelial. Cells were collected at the lowest possible passage after cloning (p3 to p8) to minimise any effects of genomic instability caused by deregulated HPV16 oncogenes.  Cells were lysed on ice for 30 minutes in 50 ml freshly prepared icecold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal CA-630, one protease inhibitor cocktail tablet (Roche complete, EDTA-free)). Nuclei were pelleted (650 g for 5 minutes at 4˚C), washed once with 1.25 x NEBuffer 2, and then resuspended in 1.25 x NEBuffer 2 to make aliquots of 5-6 million cells for digestion. SDS was added (0.3% final concentration) and the nuclei were incubated at 37˚C for one hour (950 rpm). Triton X-100 was added to a final concentration of 1.7 % and the nuclei were incubated at 37˚C for one hour (950 rpm). Restriction digest was performed overnight at 37˚C (950 rpm) using 800 units MboI (NEB) per 5 million cells. Restriction fragment ends were filled in using Klenow (NEB) with dCTP, dGTP, dTTP and biotin-14-dATP, and the blunt-ended DNA was ligated following an in-nucleus ligation protocol.  Excess salts and enzymes were removed by centrifugation (600 g for 5 minutes at 4˚C) and the cell pellet was re-suspended in 995 µl of 1x ligation buffer (NEB) supplemented with BSA (100 µg/mL final concentration). Ligation was carried out using 2000 units of T4 DNA ligase (NEB) per 5 Mio starting material of cells, at 16˚C for 4 hours, followed by 30 min at room temperature.  Chromatin was then decrosslinked overnight at 65˚C in the presence of proteinase K (Roche), purified by phenol and phenol-chloroform extractions, precipitated with ethanol and sodium acetate and re-suspended in TLE (10 mM Tris-HCl pH 8.0; 0.1 mM EDTA). 40 µg of Hi-C library DNA were incubated with T4 DNA polymerase (NEB) for 4 hours at 20˚C to remove of biotin from nonligated fragment ends, followed by phenol/chloroform purification and DNA precipitation overnight at -20˚C. DNA was sheared to an average size of 400 bp using the Covaris E220 (settings: duty factor: 10%; peak incident power: 140W; cycles per burst: 200; time: 55 seconds). End-repairing of the sheared DNA (using T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB)) was followed by dATP addition (Klenow exo-, NEB) and a double-sided size selection using AMPure XP beads (Beckman Coulter) to isolate DNA ranging from 250 to 550 bp. Biotin-marked ligation junctions were immobilised using MyOne Streptavidin C1 Dynabeads (Invitrogen) in binding buffer (5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1M NaCl) and after stringent washing in the same buffer at 55°C for 10 min ligated to the custom SCRiBL adapter using 1600 units of T4 DNA ligase (NEB) for 2 hours at room temperature. These adapters were generated by annealing SCRiBL_adapter_1 and SCRiBL_adapter_2 (table X). The immobilised Hi-C libraries were amplified using the custom primers PE PCR 1.0.33 and PE PCR 2.0.33 with 7-9 cycles. After PCR amplification, the HiC libraries were purified with AMPure XP beads (Beckman Coulter). Quantity and integrity of the Hi-C libraries was determined by Bioanalyzer profiles (Agilent Technologies).