Comprehensive Long Span Paired-End-Tag Mapping Reveals Recurrent Fusion Genes in Gastric Cancer Genomes
ChIA-PET sample preparation was performed as described earlier (Fullwood et al., 2010) with modifications of the library construction. After chromatin was precipitated using RNA polymerase II antibodies, DNA fragments present in chromatin fragments were end-repaired using T4 DNA polymerase (NEB), followed by overnight ligation at 16°C with barcoded LMP CAP half-linkers A and B containing only a single 5’ phosphorylated end to the end-repaired DNA using T4 DNA ligase (Fermentas). The chromatin was then eluted off the beads with 1% SDS, followed by Triton X-100, and ligated overnight at 16°C under ultra-dilute conditions (< 0.01 ng DNA per µl reaction) with a biotinylated sticky internal adaptor, creating a nick on each strand of DNA. The cross-links in the DNA/protein complexes were reversed by incubation at 50°C for 2 hours with proteinase K (Ambion), and the DNA fragments were purified by phenol/chloroform extraction and isopropanol precipitation. Both nicks were translated >50 bp into the circularized genomic DNA fragment by DNA polymerase I (NEB), and paired-end tags of >50 bp were released by T7 exonuclease (NEB) and S1 nuclease (Life Technologies). The digested, biotinylated PETs were then immobilized on streptavidin-conjugated magnetic M-280 Dynabeads (Life Technologies) and the ends of each PET structure were then ligated with Illumina PE adapters by T4 DNA ligase (Fermentas) at 16°C overnight with mixing followed by 16 rounds of PCR to amplify the PETs. The library was sequenced by 2 X 101 on an Illumina HiSeq 2000 Sequencer.