Serum HBV DNA quantification

Submandibular bleeds (50–75 μl) were taken from mice once weekly after induction of infection for 8 weeks. The blood was allowed to clot and then the serum was separated from plasma by two rounds of centrifugation at 6000×g for 10 and 2 min, respectively, at room temperature. Viral DNA was extracted from 40 μl of serum using the Invisorb Virus DNA HTS 96 kit (Stratec, Birkenfield, Germany) according to manufacturer’s protocol and isolated DNA was eluted in 40 μl of elution buffer. HBV-DNA serum levels were quantified by quantitative real-time PCR using the FastStart Universal SYBR Green Master (Rox) Kit (Roche, Basel, Switzerland) on a LightCycler 480 II Machine (Roche) with absolute quantification software (Roche). A serial dilution of the linear HBV plasmid was used as internal standard, HBV D3 was linearized with PstI (New England Biolabs), HBV C2 with HindIII (New England Biolabs), and HBV-A2 was linearized with SapI. The limit of detection of serum HBV-DNA is 500 copies/ml.