GSM9537685: AAV2_Rep1; Homo sapiens; ChIP-Seq
For Viral Chromosome Conformation Capture assay, wtAAV2-infected cells at 24 hpi were crosslinked with 1% formaldehyde for 10 minutes, quenched with 0.125 M glycine, lysed in NP-40 lysis buffer for 10 minutes on ice, before resuspending the nuclei in restriction enzyme buffer (Buffer 2.1). Samples were permeabilized in 0.3% SDS for 1 hour on a 37 degree C shaker, sequestered in 2% Triton X-100 for 1 hour on a 37 degree C shaker. Pellet was resuspended in restriction enzyme buffer (Buffer 2.1) and digested overnight in 400 U of HindIII enzyme. Digestion was continued for 4 more hours next day with 300 U more HindIII enzyme. The restriction enzyme was inactivated with SDS and heat for 20 mins, and samples were subjected to intramolecular ligation with T4 DNA Ligase at room temperature for 4 hours. Samples were then reverse-crosslinked and purified before digestion with the secondary restriction enzyme NlaIII (NEB). NlaIII was heat-inactivated, samples were purified using phenol: chloroform, 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 wtAAV2 genome. 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 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 and submitted for library preparation and high-throughput sequencing. Libraries were prepared from 1 microgram of DNA using NEBNext Ultra II DNA Library Prep Kit and sequenced using single-end sequencing on an Illumina NextSeq platform spiked with 25% phiX spike-in to increase library complexity. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (cat # E7645L) according to the manufacturer's instructions. Six samples were pooled per run and samples were analyzed by 75 bp paired-end sequencing on the NextSeq 500 (Illumina).