Chromatin immunoprecipitation (ChIP)-reChIP assays

ChIP-reChIP assays were performed by Re-ChIP-IT kit (Active Motif, Cat# 53016) following the manufacturer’s recommendations. Briefly, after first ChIP with P65 antibody (CST, Cat#8242), the protein-chromatin complex was divided equally into several aliquots. Samples not used for second ChIP were decrosslinked and purified by Proteinase K and phenol/chloroform extractions. The other aliquot of chromatin was subsequently used in the second ChIP reaction. Chromatin was washed, then eluted with a specific Re-ChIP-IT elution buffer. Next, chromatin from the first ChIP reaction was desalted and subjected to second ChIP with Snail antibody (R&D, Cat#AF3639). In addition, a control sequential ChIP was carried out in which chromatin was immunoprecipitated first with an antibody against P65 followed by no antibody. Aliquots of input DNA before and after performing the first ChIP reaction were used as controls in PCR analysis together with the second ChIP samples. As a negative control, rabbit IgG was used for non-specific ChIP. Re-ChIP chromatin level was determined by semi-quantitative PCR analysis. The PCR products were separated on a 1% agarose gel containing ethidium bromide for visualization and analysis.