Download ENCODE Snyder lab ChIA-PET protocol (3-1

ENCODE Snyder lab ChIA-PET protocol (3-1-16)
Fixation: Cells were crosslinked with formaldehyde at a final concentration
of 1% for 10 minutes at room temperature. The reaction was quenched with
Glycine at a final concentration of 125mM and nuclear lysates were
sonicated using a Branson 250 Sonifier (power setting 2, 100% duty cycle
for 7 x 30-s intervals).
ChIA-PET protocol: We followed the published protocol by (Heidari et al.,
2014) with modifications as described in detail here. Briefly, cells were
crosslinked and subjected to nuclear lysis followed by chromatin shearing.
Immunoprecipitation was performed overnight at 4 °C with antibodies
against the cohesin subunit Rad21. The antibody used for ChIA-PET is
Abcam Anti-Rad21 antibody (ab992). The immuno-complexes were pulled
down with Protein-G dynabeads (Lifetechnologies #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 use of Proteinase K followed by DNA purification. DNA fragments
were tagmented with Illumina nextera transposase. After Streptavidin pulldown fragments were amplified using PCR and subjected to Illumina
sequencing.