2.9. cccDNA Extraction

HBV cccDNA was extracted using the Hirt protein-free DNA extraction procedure, as described previously [45]. Briefly, 3 × 10⁶ HepG2, HepG2-hNTCP-C9, and HepG2-hNTCP-C9-HA-CaMKII α, or -Myc-AMPK α1 cells in collagen-coated 10 cm dishes were infected with HBV as described above. Seven days after infection, the cells were lysed with 0.6% SDS-TE buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA) for 30 min at room temperature. The NaCl concentrations of the lysates were adjusted to 1 M with 5 M NaCl, and the lysates were incubated at 4 °C for 16 h, and centrifuged at 14,500× g for 30 min. The supernatants containing cccDNA were extracted twice with phenol and once with phenol-chloroform, followed by ethanol precipitation, with cccDNA quantified by Southern blotting. To further validate the authenticity of HBV cccDNA, the Hirt DNA sample was heated to 85 °C for 5 min. Among three forms of HBV DNA from the Hirt protein-free DNA extraction sample, including cccDNA (2.1 Kbp), DL DNA (3.2 Kbp), and protein-free RC DNA (above 3.2 Kbp), RC and DL DNAs were denatured but cccDNA was not. Electrophoretic mobility of cccDNA remains unchanged. Then, heat-treated DNA sample was further digested with EcoR I to linearize cccDNA to a genome-length double-stranded DNA (3.2 Kbp).