Hi-C of homo sapiens (human) liver cells (PHH) of patient 399, infected with HBV, infected with HBV (BC176), replicate 2
Hi-C library generation was carried out as described previously 4 with minor modifications. Five millions PHH cells were collected and incubated for 10 min at RT in culture medium containing 1% formaldehyde. The reaction was stopped by addition of 2.5M glycine (final concentration: 0.2M) and incubated for 5 min at RT followed by 15 min on ice. The suspension of crosslinked cell was centrifuged at 300g for 10 min, and the pellet suspended in 500l ice-cold Hi-C lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630) with 100l of protease inhibitors (Sigma, P8340). The suspension was incubated on ice for 15 min, centrifuged (2500g for 5 min), and the cell pellet suspended in 1ml of digestion buffer 1X (NEB). The cells were transferred in a 2mL tube Soft tissue homogenizing CK14 (BERTIN Instruments), then lysed using tissue homogenizer (Precellys 24, BERTIN Instruments; 6,700 rpm, 3 cycles 20s on/30s off, repeated twice). Lysate was transferred into loBind tubes (Eppendorf). Precellys beads were washed with 800l NEB buffer subsequently pooled with lysate. Total lysate was split into six 300l aliquots. Each aliquot was mixed with 200l NEB buffer 1X, followed by the addition of 15l SDS 10% and incubation at 65C for 10 min. SDS was quenched by adding 100l of 10% Triton X-100 in each tubes which were kept at 37C for 30 min. Chromatin was digested overnight at 37C by adding 1000 Units of BglII (NEB). DNA ends were filed and labeled with biotin in klenow end-filling reaction with 1.5l of 10mM dATP, 1.5l of 10mM dGTP 1.5l of 10mM dTTP, 37,5ul of 0.4mM biotin-14-dCTP (Life Technologies, 19518018) and 40 U of Klenow (NEB, M0210) incubated at 37C for 45 min. Digested DNA was then ligated by adding 900l of ligation buffer containing 120l of 10X NEB T4 DNA ligase buffer, 100l of 10% Triton X-100, 12l of 10mg/ml Bovine Serum Albumin, 663l of water and 5l of 400 U/ l T4 DNA Ligase (NEB, M0202) and incubation at RT for 4 hours under slow rotation. After crosslink reversal and protein degradation with proteinase K (65C, overnight), DNA was purified using phenol-chloroform extraction and suspended in TE 1X supplemented with RNase A (10 g/mL final). DNA was then sheared using a Covaris LE220 apparatus (Duty cycle 15, intensity 150, Cycles/burst 200, time 80 secs). After shearing DNA was run on 1% agarose gel and bands ranging from 300 to 800 pb were extracted from the gel using minielute gel extraction kit (Qiagen). DNA fragments were immobilized using MyOne Streptavidin C1 dynabeads (Life Technologies), end repaired, adenine tailed and ligated to paired-end adaptors (illunina). The immobilized Hi-C DNA was amplified using Illumina primers PE PCR 1.0 and PE PCR 2.0 with 8 to 12 PCR amplification cycles. PCR products were further used for direct Illumina pair-end sequencing using nextSeq 500 High output kit v2 (Illumina), or processed through an additional step of capture C (CHi-C).