ChIA-PET Protocol with Chromatin Shearing

We performed ChIA-PET experiments with modifications to previously published protocols^{2 (link),22 (link)}. These modifications have also been independently described^{17 (link),23 (link)}. We used Illumina’s Nextera tagmentation to generate sequencing libraries. In brief, cells were crosslinked and subjected to nuclear lysis followed by chromatin shearing (no restriction enzyme was used). Immunoprecipitation was performed overnight at 4 °C with antibodies against the cohesin subunit RAD21 (Abcam Anti-RAD21 antibody (ab992) https://www.encodeproject.org/antibodies/ENCAB529YRC/). The immuno-complexes were pulled down with Protein-G dynabeads (Life Technologies #10003D, New York). Biotinylated linkers were ligated to the enriched fragments, followed by proximity ligation overnight at 16 °C.
Crosslinking was reversed at 65 °C with the use of Proteinase K followed by DNA purification. We used Illumina Nextera Transposase to add sequencing adapters to ChIA-PET libraries. Biotinylated fragments were enriched by pull-down with Streptavidin Dynabeads (M-280; Lifetechnologies #11205D, New York). The final libraries were sequenced on an Illumina HiSeq 2000.