title: GSM5979893: 3-2; Homo sapiens; Hepatitis B virus; ChIP-Seq
library_construction_protocol: The cccDNA ChIP-Seq assay was performed according to previously described procedures, with slightly modifications. Huh7 cells transfected HBVcircle were cross-linked by 1% formaldehyde (Invitrogen, USA) for 10 min at room temperature, and fixation was quenched by adding 125 mM glycine final concentration for 5 min. Next, the cells were permeated in nuclei isolation buffer [1× PBS with 0.1% Triton X-100, 0.1% NP-40, 1 mM DTT, 10 mM sodium butyrate, 1× protease inhibitor (Roche)] for 10 min at 4℃. Nuclei were pelleted after centrifugation at 9000g for 3 min at 4℃, resuspended and digested in Mnase digestion buffer [50 mM Tris pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 10% (vol/vol) glycerol, 10 mM sodium butyrate, 1× protease inhibitor] with 1500 U/mL Mnase (New England Biolabs, Ipswich, Massachusetts) at 37℃ for 1 h. and digestion was quenched with 10 mM EGTA final concentration on ice. Then, the digested nuclei were harvested by centrifugation at 9000g for 3 min at 4℃, and resuspended by ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 10 mM sodium butyrate, protease inhibitor) with occasional mixing for 2 h at 4℃. The sample was centrifugated at 6500g for 5 min, and the supernatant was collected for immunoprecipitation. For ChIP, the above lysis supernatant was added into protein A/G-conjugated agarose beads after protein A/G-conjugated agarose beads was incubated with indicated antibodies in a rotator overnight at 4℃, and followed by collecting beads and washing five times. Immunocomplexes was eluted in elution buffer (1% SDS, 100 mM NaHCO3), and decross-linked at 65 °C for 5–6 h, the DNA fragments were purified by a TIANamp Genomic DNA Kit (TIANGEN Biotech Co., Beijing, China). For sequencing, the DNA fragments were sent to sequencing company (Annoroad Genomics Company, China). Repair the ends of DNA fragments, add A to the end of the DNA fragment, adaptors were added into the end of the DNA fragments, PCR amplification,  DNA fragments purification.