Instrument: Illumina MiSeq
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were fixed in freshly prepared 1% formaldehyde in PBS (both ThermoFisher Scientific) for 5 min prior to quenching with 125 mM glycine in PBS. Pelleted cells were either used directly for analysis or snap-frozen and stored at -80°C until use. Next, cells were washed with cold lysis buffer [PBS with 0.1% Triton X-100, 0.1% NP-40, 1 mM dithiothreitol (DTT), 50 ng/ml trichostatin A (TSA) and 1x EDTA-free protease inhibitor (Roche, Basel, Switzerland)] and lysed in the same buffer for 10 min on ice. Nuclei were pelleted, resuspended in digestion buffer [H2O with 50 mM Tris-HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 10% glycerol, 50 ng/ml TSA, and 1x EDTA-free protease inhibitor (Roche)] and digested with 600 IU/ml micrococcal nuclease (MNase; ThermoFisher Scientific) for 12 min at 37°C. Digestion was stopped by addition of 10 mM EDTA. Nuclei were pelleted at 6500 x g and supernatants collected. The pellet was resuspended in digestion buffer with 10 mM EDTA and 300 mM NaCl and mildly sonicated using a W 375 sonicator (Qsonica, Newtown, CT) at 50% duty cycle and power setting 3. Nuclei were again pelleted, supernatants combined and mixed with an equal amount of sucrose buffer [H2O with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.01% NP-40, 50 ng/ml TSA, and 1x EDTA-free protease inhibitor (Roche)]. Samples were concentrated using Amicon Ultra-4 100 kDa centrifugal filter units (Millipore-Sigma; Merck KGaA, Darmstadt, Germany) and spun on a 5-30% continuous sucrose gradient in sucrose buffer for 4 h at 40,000 x g and 4°C using a SW41Ti rotor (Beckman Coulter Inc., Brea, CA). Chemicals were obtained from Millipore-Sigma unless noted otherwise. Mononucleosome containing fractions were pooled and concentrated to ~500 µl prior to addition of 100 ng/µl bovine serum albumin (ThermoFisher Scientific). Mononucleosomes were either stored at -20°C or used immediately for ChIP. Antibodies were bound to 20 µl/ChIP Dynabeads Protein G (ThermoFisher) in ChIP buffer [H2O with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 200 µg/ml BSA] at 4°C and added to 2 µg of chromatin in ChIP buffer and allowed to bind overnight. Samples were washed six times in LiCl wash buffer [H2O with 150 mM LiCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, and 0.7% sodium deoxycholate] and eluted in 100 µl elution buffer [H2O with 1% SDS and 100 mM NaHCO3]. Crosslinks were reversed by digestion with Proteinase K (ThermoFisher) for 5 h at 65°C and then digested for another 30 min with DNase-free RNase A (ThermoFisher) at 37°C. DNA was purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 1 ng of DNA was used for sequencing library construction with the KAPA Hyper Prep Kit (Roche) according to the manufacturer's instructions. For barcoding, 2 µl of TruSeq RNA adapters stocks were used (Illumina, Inc., San Diego, CA). Up to 6 libraries were pooled per sequencing run and 500 ng pooled library subject to HBV-specific target enrichment using the xGen Lockdown Reagents Hybridization and Wash Kit (Integrated DNA technologies, Skokie, IL) according to the manufacturer's instructions. A custom set of xGen lockdown probes of 60 bp length tiling the entire HBV genome of genotypes A-D was used for target enrichment (Integrated DNA technologies) together with Dynabeads MyOne T-270 Streptavidin (ThermoFisher). The pull-down was amplified for 12 or 8 PCR cycles (biopsies vs. de novo infected cells/cell lines; KAPA HiFi HotStart ReadyMix, Roche) and the product cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Inc.). The product was used to dilute the original library to 20 nM. Sequencing was performed using an Illumina MiSeq sequencer with a MiSeq v3 reagent kit for 2x76 bp paired-end reads (Illumina, Inc.).