EXPERIMENT_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
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 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.  Capture RNA bait library design 120-mer capture RNA baits were bioinformatically designed to both ends of MboI restriction fragments overlapping the HPV16 genome. Requirements for target sequences were as follows: GC content between 25% and 65%, no more than two consecutive Ns within the target sequences, and maximum distance to a MboI restriction site 330 bp. For short MboI fragments, where 120-mer RNA baits originating from both ends would have overlapped (potentially interfering with optimal hybridization to Hi-C libraries), only the Watson (coding or sense) strand was used for capture RNA bait design, and if necessary the baits were trimmed to minimum length no shorter than 97 nt. This yielded 16 RNA bait sequences covering the MboI restriction fragment ends of the entire HPV16 genome, with the exception of two fragments too short (18 and 63 bp, respectively) for capture RNA bait design. 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).  Solution hybridization Region Capture of Hi-C libraries. 500 ng of Hi-C 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).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Two biological replicates Hi-C and HPV16-specific Region Capture Hi-C libraries were prepared for each of the W12 cell lines. Capture Hi-C was performed using HPV16-specific RNA baits. Hi-C libraries alone were prepared from normal cervix tissue (Ncx).
SAMPLE_TITLE: W12 Clone G2 cHi-C rep II
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "ENA first public", "VALUE": "2021-03-07"}, {"TAG": "ENA last update", "VALUE": "2021-06-18"}, {"TAG": "External Id", "VALUE": "SAMEA8139041"}, {"TAG": "INSDC center alias", "VALUE": "EMBL-EBI"}, {"TAG": "INSDC center name", "VALUE": "EMBL-EBI"}, {"TAG": "INSDC first public", "VALUE": "2021-03-07T04:48:17Z"}, {"TAG": "INSDC last update", "VALUE": "2021-06-18T16:17:05Z"}, {"TAG": "INSDC status", "VALUE": "public"}, {"TAG": "Submitter Id", "VALUE": "E-MTAB-10152:W12 Clone G2 cHi-C rep II"}, {"TAG": "broker name", "VALUE": "ArrayExpress"}, {"TAG": "cell line", "VALUE": "W12"}, {"TAG": "cell type", "VALUE": "epithelial cell"}, {"TAG": "common name", "VALUE": "human"}, {"TAG": "disease", "VALUE": "squamous cell neoplasm"}, {"TAG": "experiment", "VALUE": "HPV capture Hi-C"}, {"TAG": "organism part", "VALUE": "cervix epithelium"}, {"TAG": "sample name", "VALUE": "E-MTAB-10152:W12 Clone G2 cHi-C rep II"}, {"TAG": "strain", "VALUE": "W12 Clone G2"}]
Pool_Member_@attributes_sample_title: W12 Clone G2 cHi-C rep II
RUN_SET_RUN_Pool_Member_@attributes_sample_title: W12 Clone G2 cHi-C rep II
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