Gel extraction kit

The DIG Northern Starter Kit (12039672910, Roche, Germany) was used in the experiments. Briefly, total cellular RNA was extracted and electrophoresed on 1.4% formaldehyde-agarose gel. The RNA was then transferred onto a nylon membrane and fixed by UV cross-linking. The nylon membrane was hybridised with a digoxigenin-labelled specific RNA probe (corresponding to nucleotides 126–1,225 of the HBV genome) overnight at 68°C. The membrane was incubated in blocking solution for 30 min and antibody solution for 30 min at 37°C. The signal was detected by exposing on an X-ray film.
For HBV RNA probe, three 500 bp HBV DNA fragments (corresponding to nucleotides 126–1,225 of the HBV genome) were amplified by PCR using pCH9/3091 plasmid as template. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit (D2111-02, Magen, China) and PCR product purification kit (11732676001, Roche, Germany). Three 500 bp HBV DNA fragments (corresponding to nucleotides 126–1,225 of the HBV genome) were labelled with digoxigenin-11-UTP with a kit supplied by Roche (12039672910). Labelling was carried out by following the manufacturers’ instructions.

The DIG-High Prime DNA Labelling and Detection Starter Kit (11585614910, Roche, Germany) was used in the experiments. The HBV core DNA and Hirt-extracted DNA samples were subjected to 1% agarose gel electrophoresis and transferred onto a nylon membrane. The nylon membrane was cross-linked by exposure to UV light in a Stratalinker UV crosslinker and hybridised with a digoxigenin-labelled HBV full-length genomic DNA probe overnight at 42°C. The membrane was incubated in blocking solution for 30 min and antibody solution for another 30 min at 37°C. The signal was detected by exposing on an X-ray film. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA.
For HBV DNA probe, full-length HBV DNA was amplified by PCR using pCH9/3091 plasmid as template. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit (D2111-02, Magen, China) and PCR product purification kit (11732676001, Roche, Germany). Full-length HBV DNA was labelled with digoxigenin-11-dUTP with a kit supplied by Roche (11585614910). Labelling was carried out by following the manufacturers’ instructions.

To sequence the genomic loci of interest, we initiated by amplifying them from genomic DNA samples using amplification primers (Supplementary Tables 5 and 6) that contained Illumina forward and reverse adapters. The specific genomic region was amplified in the first round of PCR (PCR 1) using these primers. In a subsequent PCR reaction (PCR 2), unique Illumina barcoded primer pairs were incorporated into each sample. The PCR2 products, obtained by pooling the common amplicons, were purified using a gel extraction kit (Magen, Guangzhou) after 1.5% agarose gel electrophoresis and eluted with 40 μL of water. The concentration of DNA was measured through fluorescence quantification or qPCR, and sequencing was performed on an Illumina NovaSeq instrument following the manufacturer’s protocol.^{43 (link),47 (link)} Annoroad Gene Technology Co., Ltd. (Beijing) conducted targeted deep sequencing, and the resulting data were analyzed using CRISPResso2 and custom R scripts.