Protein a g conjugated agarose beads

ChIP assay was performed as described previously.⁴⁶ (link) Briefly, 50-mg liver tissues were minced by Tissue Cell-Destroyer (DS1000; Novastar) and homogenized in PBS and fixed in 1% formaldehyde (Invitrogen) for 10 minutes at room temperature, and 1/20 volume of 2.5 M glycine (Sigma-Aldrich) was added to quench formaldehyde. Then, ChIP lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor) was added and samples were kept on ice for 30 minutes. The lysates were sheared by sonication using a Bioruptor Plus (Diagenode, Seraing, Belgium). Crosslinked chromatin samples were incubated with indicated antibody or normal rabbit immunoglobulin G in a rotator overnight at 4°C. Subsequently, protein A/G-conjugated agarose beads (Smart-Lifesciences, Changzhou, China) were added and incubated in a rotator overnight at 4°C, then the beads were collected and washed 3 times. To elute DNA fragments, immunocomplexes were incubated with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1.0% SDS) for 2 hours at 65°C. The elusion was treated with proteinase K (Sigma-Aldrich) overnight at 55°C. Finally, the DNA was purified with a TIANamp Genomic DNA Kit (TIANGEN Biotech, Beijing, China). The purified DNA was detected by qPCR amplification with primers specific for cccDNA. The information of antibodies used is listed in Table 2.