title: Illumina HiSeq 2500 paired end sequencing; Targeted capture DNA-seq for HPV16-human breakpoint junctions in HPV16 infected W12 cervical cell lines
design: Targeted capture DNA-seq for HPV16-human breakpoint junctions in HPV16 infected W12 cervical cell lines
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 byderegulated HPV16 oncogenes. Biotinylated RNA bait library generation for HISC Four consecutive and non-overlapping fragments from the pSP64 HPV16 bacterial artificial chromosome were PCR amplified using the Expand High Fidelity PCR system (Roche).  This resulted in complete coverage of the HPV16 genome.  T7 promoter sequences (Roche) were added to one side of the PCR product during this PCR amplification, enabling subsequent directional in vitro transcription.  Sequences were in vitro transcribed in the presence of biotinUTP and purified as described above, followed by fragmentation to 120 nt with 4 nM magnesium chloride at 95°C for 7 min.  Fragmented biotinylated RNA was purified by Isopropanol precipitation followed by two subsequent washes with 75% ethanol.  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. 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).  Ligation of the custom sequence adapters was done in solution by adding 4 microlitre adaptors (30 micromolar) and 1600 units T4 DNA ligase (NEB).  The ligation was carried at for 2 hours at room temperature on a rotating wheel in 1x ligation buffer (NEB). Pre-capture PCR amplification was carried out using the custom primers PE PCR 1.0.33 and PE PCR 2.0.33 with 7-8 cycles.  The amplified libraries were purified with AMPure XP beads (Beckman Coulter) and the quantity and the quality was assessed by Bioanalyzer profiles (Agilent Technologies).  500 ng of genomic library DNA were concentrated using a vacuum concentrator (Savant SPD 2010, Thermo Scientific) and then re-suspended in 5 microlitre dH2O. 2.5 microgram mouse cot-1 DNA (Invitrogen) and 2.5 microgram sheared salmon sperm DNA (Ambion) were added as blocking agents. To prevent concatemer formation during hybridization, 1.5 microlitre blocking mix (300 mircomoles) was added (equimolar mix of P5_b1_for_33, P5_b1_rev_33, P7_b2_for and P7B2_rev). Biotinylated RNA baits were used in a ratio 1:12 to Hi-C libraries (25 ng biotinylated RNA baits per 300 ng of Hi-C library) and supplemented with 30 units SUPERase-In (Ambion). The DNA was denatured at 95˚C for 5 min in a PCR machine (PTC-200, MJ Research; PCR strip tubes (Agilent 410022)) and then incubated with the biotin capture RNA at 65˚C, in hybridization buffer (5 x SSPE (Gibco), 5 x Denhardt's solution (Invitrogen), 5 mM EDTA (Gibco), 0.1 % SDS (Promega)) for 24 hours, in a total reaction volume of 30 microlitre. Captured DNA/RNA hybrids were enriched using DynabeadsMyOne Streptavidin T1 beads (Life Technologies) in binding buffer (1 M NaCl, 10 mM TrisHCl pH 7.5, 1 mM EDTA) for 30 minutes at room temperature. After washing (once in washbuffer 1 (1 x SSC, 0.1 % SDS) for 15 minutes at room temperature, followed by three washes in wash buffer 2 (0.1 x SSC, 0.1 % SDS) for 10 minutes each at 65˚C), the streptavidin beads(with bound captured DNA/RNA) were re-suspended in 30 microlitre 1 x NEBuffer 2. Post-capture PCR amplification was carried out using between six to nine cycles using primer pairs that consisted of one TruSeq adapter reverse compliment and the TruSeq universal adapter from streptavidin beads in multiple parallel reactions, which were then pooled to purify the PCR products using AMPure XP beads (Beckman Coulter).