Preparation of viral DNA and quantitative PCR analysis

The sequence and position of all primers used for qPCR and RT-qPCR is indicated in Table 1.

For quantification of HBV copy numbers in supernatant of HepG2.2.15 cells or HBV infected HepG2-NTCP cells, viral DNA was isolated using the High Pure Viral Nucleic Acid Kit (ROCHE diagnostics) according to manufacturer’s protocol.

Purified DNA was used for real time PCR with Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and HBV DNA specific primers (HBV RC F; HBV RC R, Table 1). Absolute copy numbers were calculated referring to a standard based on the linearized HBV coding plasmid pJO19.

For analysis of intracellular total HBV DNA and cccDNA from infected HepG2-NTCP cells, DNA from total cell lysates was prepared using the NucleoSpin Tissue Kit (Macherey-Nagel 740952.250) according to manufacturer’s protocol with minor modifications. Briefly, cells were trypsinized and re-suspended in provided T1 buffer and incubated with proteinase K and B3 buffer at 70 °C for 1 hour and following steps were stick to the protocol. For cytosolic HBV DNA in HepG2.2.15 cells, cells were treated with fractionation buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl) containing 1% NP-40. NP-40 soluble fraction was transferred and mixed with proteinase K and B3 buffer and cytosolic DNA was obtained using the same kit. For cccDNA quantification, DNA samples were digested with T5 exonuclease (New England Biolabs. M0363). Products were subjected for qPCR with cccDNA specific primers spanning the gap region). This protocol was shown to specifically allow the detection of cccDNA and to avoid contamination by rcDNA⁷⁰ (Qu et al., manuscript in preparation). β-globin expression levels from undigested samples were used for normalization (β-globin-F; β-globin-R). For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles). A plasmid carrying two copies of full-length HBV genome was used for standard curve⁷¹.