ChIP re-ChIP experiments were performed as described previously (Wienerroither et al., 2014).
To directly test whether RNAPII and PRCs simultaneously coassociate at PRC-repressed chromatin, we used sequential ChIP (re-ChIP).
Results from sequential ChIP (re-ChIP) show that the two complexes have similar accessory-factor compositions but differ in their preference for the recruitment of coactivators and corepressors.
bp: base pair; CEAS: the Cis-regulatory Element Annotation System; ChIP: chromatin immunoprecipitation; DFX: deferroxamine; MEF: mouse embryo fibroblast; miRNA: microRNA; NF: nuclear factor; NFI: NFκB activation inhibitor; qPCR: quantitative PCR; re-ChIP: sequential ChIP.
ChIP and RE-ChIP analyses of DMR1 sequence support the proposition of co-occupancy by Ctcf, Parp1, PARs and Dnmt1.
To relate the gene activation with the direct binding of p53 to its BAX-RE or the NOS3-RE, ChIP assay was conducted in H9c2 cells.
In order to correlate the gene activation with the direct binding of p53 to its BAX-RE or the NOS3-RE, ChIP assay was conducted in RNC, RNC p53−/− and RNC p53-Lys(Mut) cells.
In RE-ChIP assays the primary ChIP (for Ctcf) was performed accordingly to the standard protocol starting from 2×10 cells for each IP.
The re-ChIP DNA was eluted with ChIP elution buffer as described in the single ChIP protocol above, and ChIP samples were analyzed by qPCR.
ChIP/QPCR and re-ChIP/QPCR were performed and analysed essentially as previously described [ 5, 7] using the ChiP-IT Express Enzymatic Kit and re-ChIP-IT Kit (Active Motif), except that antibodies directed against Rnf2, H2AK119ub1 and H3K27me3 were used.
ChIP with phosphoacetylated (S10phK9ac) or K4me3-specific antibodies and re-ChIP experiments showed that all three modifications can occur on the same nucleosomes (Hazzalin and Mahadevan, 2005).
