Quantitative Analysis of Extracellular HBV Titers

The extracellular HBV titers were measured by quantitative real-time PCR (qPCR) as described previously [31 (link)]. Briefly, HBV genomic DNA was purified from the culture supernatant using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). For conventional PCR analysis of HBV DNA, genomic DNA was amplified using 2× Τaq PCR Master mix 1 (BioFACT, Daejeon, Korea) and a primer pair, HBV 1399F (5′-TGG TAC CTG CGC GGG ACG TCC TT-3′) and HBV 1632R (5′-AGC TAG CGT TCA CGG TGG TCT CC-3′), as described previously [24 (link)]. Additionally, qPCR for HBV was performed as previously described [31 (link)]. Briefly, HBV DNA was amplified using SYBR premix Ex Taq II (Takara Bio, Kusatsu, Japan) and a primer pair, HBV 379F (5′-GTG TCT GCG TTT TAT CA-3′) and HBV 476R (5′-GAC AAA CGG GCA ACA TAC CTT-3′), using a Rotor Gene qPCR machine (Qiagen).