Digested samples were purified, diluted and then proximity-ligated at 16☌ using T4 DNA ligase (Roche). Digestion efficiencies were quantified by qPCR using a Corbett Rotor-Gene 6000 qPCR device and Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) and primers covering the SpeI restriction site within the cccDNA normalized to a non-digested region. Prior to the primary digestion step SDS was sequestered using 2% Triton X-100, and subsequently SpeI (NEB, 600U, 24 hrs) was added. Briefly, interacting DNA segments were crosslinked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37☌ in presence of 0.3% SDS. 4C targeting on HBV cccDNA (verified genotype D3) as bait was conducted as described previously with slight modifications. 590 bp amplicon (depending on the genotype) as described above and Sanger sequencing of amplicons cloned into pGEM-T easy (Promega) via the standard T7 sequencing primer.
The genotype HBV D3, which is replicated by HepG2.2.15 cells and used for the infection of HepaRG cells, was verified by PCR using primers giving rise to an approx. In order to select a restriction strategy applicable for multiple HBV genotypes, we performed alignments using ClustalW included in molecular evolutionary genetics analysis (MEGA) 4.1. GEO help: Mouse over screen elements for information.
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