GSM8896994: 6E cells, integrated HPV31, biol rep 3; Homo sapiens; OTHER
Cells were lysed using 0.2-0.5% Igepal CA-630, centrifuged at 3,000 x g for 5 min at 4°C and the resulting nuclei permeabilized in 0.3-0.7% SDS for 1 hour at 37°C, with shaking at 900 rpm, followed by sequestration of SDS by 2% Triton X-100 for 1 hour at 37°C, 900 rpm. Nuclei were washed twice with 1X EcoRI buffer (NEB; 100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 0.025% Triton X-100, pH 7.5 @ 25°C) to remove SDS and digested overnight with 400U EcoRI (NEB, R3101M) at 37°C, with shaking at 900 rpm. An additional 300U EcoRI was added and the nuclei incubated for a further 4 hours prior to heat inactivation at 65°C for 20 min. Digested samples were ligated overnight in 7 ml 1X ligase buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) containing 400 cohesive-end units (CEU) T4 DNA ligase (NEB, M0202L) at 16°C, 500 rpm, and crosslinks reversed overnight at 56°C, 500 rpm, in the presence of 0.04 mg/ml proteinase K. Samples were then incubated with 0.04 mg/ml RNase A for 30 min at 37°C, 500 rpm. For sample S14, the primary ligation was performed overnight at 16°C, 1,400 rpm, in 50 µl 1X ligase buffer (NEB), supplemented with 0.1% Triton X-100 and 670 CEU T4 DNA ligase (NEB, M0202L), and crosslinks reversed overnight at 56°C in the presence of 0.2 mg/ml proteinase K. Samples were then incubated with 0.3 mg/ml RNase A for 30 min at 37°C. DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1, v/v; Invitrogen, 15593-031) and chloroform (Macron Fine Chemicals) extractions  and digested with 150U DpnII (NEB, R0543M) overnight at 37°C, with shaking at 900 rpm, prior to heat inactivation at 65°C for 20 min. Digested DNA samples were ligated and purified by phenol:chloroform and chloroform extractions, followed by and additional purification step using the QIAquick PCR Purification Kit (QIAGEN, 28106). Purified DNA was subjected to inverse PCR using the Expand Long Template PCR System (Roche, 11681842001) and primers that target three viewpoints within the HPV31 genome. PCR products for the three viewpoints were pooled for each sample, purified using the QIAquick PCR Purification Kit (QIAGEN, 28106) and subjected to 2 x 100 bp paired-end read sequencing to a sequencing depth of >387 million read pairs per sample. Sequencing libraries were generated using the Truseq DNA Nano kit (Illumina) and subjected to size selection to remove fragments >1,000 bp that typically represent undigested or self-ligated products. To ensure high sequence diversity of the 4C-seq libraries, a PhiX spike-in control of 30% the sequencing run was included.