Sequential ChIP-seq Protocol

Tissue fixing, chromatin sonication, and IP with the first antibody in the primary ChIP procedure were performed as described in eChIP. The first antibody was crosslinked to the beads using the fixative disuccinimidyl suberate (DSS, Pierce, 21555). After washing with low-salt ChIP buffer, high-salt ChIP buffer, ChIP wash buffer, and TE buffer, the protein-DNA complexes were eluted from beads by ChIP Elution buffer for 15 min at 65 °C with agitation at 900 rpm. The supernatant (eluted chromatin) was then transferred to a new tube. A total 5% of the eluted chromatin was retained for the primary ChIP DNA purification. The remaining eluted chromatin was then used for the reChIP. reChIP with the secondary antibody, elution, DNA purification, library construction, and sequencing were performed as described above.