The agarose beads were washed, chromatin extracted and protein-DNA cross-links reversed.
The samples were then washed, eluted in SDS Elution Buffer, and the cross-links reversed by overnight incubation at 65°C.
The transcription factor-DNA complexes were enriched by chromatin immunoprecipitation, the cross-links reversed, and enriched DNA fragments and control genomic DNA fragments amplified using ligation-mediated PCR.
Protein-DNA complexes were eluted in 200 µl of elution buffer and the cross-links reversed by overnight incubation at 65°C.
DNA/protein/anti-body complexes were eluted from the beads with elution buffer (1% SDS, 0.1M NandO3) and the DNA/protein cross-links reversed with the addition of NaCl to a final concentration of 100 mM and incubation at 65°C for four hours.
The cross-link reversed DNAs were PCR-amplified using the two primer pairs shown in Figure 5A.
After six washing steps, complexes were eluted and the cross-links reversed.
Eluates were adjusted with NaCl (0.2 M) and cross-links reversed by heating the samples at 65°C overnight.
After centrifugation at 4,000 rpm for 5 min at room temperature, the two supernatant fractions were then recovered, pooled, and the DNA protein cross-links reversed with 5 M NaCl overnight at 65°C.
Antibody bound proteins were isolated on protein A agarose beads (RepliGen, Waltham, CA) or protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA), washed extensively, eluted, and cross-links reversed according to the Upstate protocol.
Complexes were captured with Protein G Dynabeads, washed with modified RIPA buffer (50 mM HEPES pH7.5, 1 mM EDTA, 0.3% Sodium deoxycholate, 1% NP40, 250 mM LiCl), eluted in 50 mM TRIS pH8, 10 mM EDTA, 1% SDS, cross-links reversed by overnight incubation at 65°C and DNA precipitated after phenol-chloroform extraction.
