Rediprime 2

Total RNA isolation was performed by vortexing cell pellets from 40 ml cultures in the presence of acid-washed baked glass beads (0.25–0.3 mm diameter) and phenol–chloroform. Isolated RNA was quantified with a NanoDrop 1000 (Thermo Scientific), separated by electrophoresis on 1.2% agarose denaturing formaldehyde gels, and blotted on to nylon membranes (Immobilon-NY+, Millipore) (Sambrook and Russell, 2001 ). Prehybridization, hybridization, and washing steps were performed following the manufacturer’s recommendations. Probes for northern blot hybridization were obtained by PCR with oligonucleotide pairs sll1815_1F/sll1815_1R for adk, sll1823_1F/sll1823_1R for purA, and sll1566_1F/sll1566_1R for ggpS (sequences listed in Supplementary Table S1) and random-prime labeled (Rediprime II, GE Healthcare) with α-[³²P] dCTP. Hybridization signals were detected and analyzed with a Cyclone Plus Storage Phosphor system (Perkin-Elmer). The signal for rnpB was used for normalization.

Total DNA and RNA were extracted from liver tissues and examined for the presence of HBV DNA and RNA by, respectively, Southern or Northern blotting as described previously17 (link). The probe was generated using a random primer labeling system (Rediprime II, GE Healthcare, Piscataway, NJ) containing a ³²P-labeled DNA fragment corresponding to the HBV small S antigen-coding sequence. Signals were visualized using a Typhoon 9410 Imager (Amersham, Bucks, UK) and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).