Anti p300

Rabbit polyclonal to H1, HMGN1, HMGN2, and H3 were from our laboratory, anti H3K27ac (Abcam#ab4729), anti H3K27me3 (Abcam#ab6002), monoclonal anti H1(Milipore-Sigma #05-457), anti CEBPB (Abcam#ab32358), Anti-Brd3 (Active Motif #61489), Anti-Brd4 (Bethyl Laboratories #A301-985A100), Anti-CEBPB (Abcam #ab32358), Anti-CTCF (EMD Millipore #07-729), Anti-Ets1 (Active Motif #39580), Anti-Ikaros (Active Motif #39355), Anti-Irf8 (Bethyl Laboratories #A304-027A), Anti-Klf4 (Abcam #106629), Anti-Nanog (Active Motif #61419), Anti-Oct4 (Abcam #ab19857), Anti-p300 (Active Motif #61401), Anti-Pax5 (Abcam #183575), Anti-Sox2 (Abcam #97959).
The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).
Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines^{55 (link)} and resting B cells^{56 (link)} were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.

Crosslink ChIP: WT-, GFP- and GN-DU145 cells (2 × 10⁷) were fixed with 1% formaldehyde (Sigma) and sonicated using the Sonifier SFX550 instrument (BRANSON, CT, USA) to induce chromatin fragmentation. Anti-NANOG (CST; #5232) and anti-p300 (Active Motif, CA, USA; #61401) Abs were bound to Protein G Dynabeads (Thermo Fisher Scientific, MA, USA), and these conjugates were used for ChIP. Native ChIP: Nuclear fractions of WT-, GFP- and GN-DU145 cells (1 × 10⁶) were treated with 1200 U MNase (Takara, Shiga, Japan) for chromatin fragmentation. The anti-H3K27Ac Ab (Abcam, Cambridge, UK; ab4729) was bound to Protein G-agarose (Merck Millipore), and the conjugate was used for ChIP. The ChIPseq library was prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and the NEBNext Multiplex Oligos for Illumina. ChIPseq libraries were sequenced by Illumina.