GSM9059593: HBV_T5_Rep1; Homo sapiens; ChIP-Seq
For Viral Chromosome Conformation Capture assays, HBV infected cells were crosslinked in 2 percent formaldehyde for 10 minutes, quenched in 0.125 M glycine for 5 minutes, collected and lysed in NP-40 lysis buffer for 10 minutes on ice, before resuspending the nuclei in restriction enzyme buffer (Cutsmart Buffer). Samples were permeabilized in 0.3% SDS for 1 hour on a 37oC shaker, sequestered in 2% Triton X-100 for 1 hour on a 37oC shaker. 1 ul of T5 Exonuclease (New England BioLabs) was added to the samples for 1 hr on a 37oC shaker. Samples were inactivated with 8 ul of 0.25 M EDTA before centrifuging at 5000 RPM for 5 minutes. Pellet was resuspended in restriction enzyme buffer (Buffer 3.1) and digested overnight in 400 U of BglII enzyme. Restriction enzyme was inactivated and samples were subjected to intramolecular ligation with T4 DNA Ligase at room temperature. Samples were then reverse crosslinked and purified, before digesting in secondary restriction enzyme, BglII. BglII was heat inactivated, samples were purified by phenol:chloroform purification, and circularized using T4 DNA Ligase. Samples were purified using a PCR Purification Kit (Qiagen), and equivalent amounts of DNA was used to perform inverse PCR with primers complementary to the HBV genome. Inverse PCR samples were diluted 1:100 in TE, before being used as templates for nested-inverse PCR reactions. 8 independent nested-inverse PCR reactions were pooled, PCR purified using a Qiagen kit and submitted for library preparation and high-throughput sequencing. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (cat # E7645L) according to manufacturer's instructions. Eight samples were pooled per run and samples were analyzed by 75 bp paired-end sequencing on the NextSeq 500 (Illumina).