Isolation of nucleic acids
Upon harvest, the cell culture medium was discarded, and the cells were
washed twice with PBS. Subsequently, the cells were lysed using
AmpliSens Riboprep lysis buffer (AmpliSense Biotechnologies). Nucleic
acids were then extracted using the AmpliSense Riboprep kit (AmpliSense
Biotechnologies) following the manufacturer’s instructions. To isolate
RNAs, the nucleic acids were treated with RNase-free DNase I (NEB) at
37°C for 30 min, purified using the AmpliSense Riboprep kit (AmpliSense
Biotechnologies), and reverse-transcribed using AmpliSens Reverta-FL
(AmpliSense Biotechnologies).
For the isolation of HBV cccDNA, the Hirt procedure, described by Caiet al .66, was used. The isolated cccDNA was
then treated with plasmid-safe ATP-dependent DNase (Epicentre) at 37°C
for 12 h, followed by enzyme inactivation at 72°C for 15 min.