Quantification of Viral Progeny by qPCR

Viral progeny was quantified by qPCR. Seventy-two hours post-transfection, cell culture supernatants were harvested and clarified by centrifugation at 3,000g for 10min. Supernatants were treated with lysis buffer (50mm Tris-HCl pH 7.5; 1mm EDTA; 1% SDS; 0.5mg/ml proteinase K (Invitrogen, United States)) and incubated at 56°C for 2h. Nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation in the presence of 20μg of dextran. qPCR was performed in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using Luna Universal qPCR Master Mix (2x; New England Biolabs, Beverly, MA, United States). The following primers were used for the amplification: sense 5’-ATGGAGACCACCGTGAACGC-3′ (nt 1608–1627) and antisense 5′-AGGCACAGCTTG GTGGCTTG-3′ (nt 1887–1868). To avoid amplification of the input linear HBV DNA used for transfection, primers that specifically amplify relaxed circular HBV DNA were used (Elizalde et al., 2019 (link)). Serial dilutions of an HBV replication-competent plasmid (pCH-9/3091) were used as quantification standards.