Total cellular RNA, cytoplasmic encapsidated HBV pgRNA, core DNA, and protein-free Hirt DNA were extracted as described previously (38 (link), 39 (link)). Non-cccDNA in Hirt DNA was removed via T5 exonuclease (New England BioLabs) digestion. EcoRI endonuclease (New England BioLabs) was then used to linearize HBV cccDNA before electrophoresis.
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.