GSM5387603: U2OS_AAV_36hpi_rep_2; Homo sapiens; ChIP-Seq
For ChIP assays, lysates were clarified from sonicated nuclei (Diagenode), and DNA-protein complexes were immunoprecipitated using antibodies conjugated to Dynabeads (ThermoScientific). For Viral Chromosome Conformation Capture assays, AAV2/rAAV 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 containing 3C Lysis buffer for 10 minutes before being subjected to primary restriction enzyme digestion overnight with 400 U of HindIII. Following digestion, the 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 the secondary restriction enzyme, NlaIII. NlaIII 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 AAV2/rAAV genome. Inverse PCR samples were diluted 1:100 in TE, before being used as templates for nested-inverse PCR reactions. 6 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. Twelve samples were pooled per run and samples were analyzed by 75 bp single-end sequencing on the NextSeq 500 (Illumina).