Anti h3k27ac

Tc17 cells were cultured for 72 h ± DMF or DMF + GSH as indicated in Figure legends. 2–5 × 106 cells were crosslinked with 1% formaldehyde for 6 min at room temperature subsequently, ChIP was performed. Lysed cells were sonicated in a Bioruptor® Plus (Diagenode) with 30s ON, 30s OFF on high power output for 27–33 cycles at 4 °C. For immunoprecipitation, 2.5–4 µg of the following Abs were used: anti-H4ac (Millipore, 06–866), anti-H3K4me3 (Active Motif, 39159), anti-H3K27me3 (Active Motif, 39155), anti-H3K27ac (Abcam, ab4729) or control IgG (Cell Signaling, 2729). Primer sequences for Il17a promoter, Il17 enhancer-5, Il10 promoter and Rpl32 are provided in Supplementary Table 5. Amplifications were performed at the ABI Prism7500 (Applied Biosystems) using the Fast SYBR™Green (Thermo Fisher, 4385610). Values for non-specific binding (determined by control IgG) were subtracted. After normalization, the specific pulldown (input %) was calculated.

To investigate the binding of CCCTC-binding factor (CTCF) to chromatin, ChIP analysis was carried out after sonicating the chromatin to an average fragment size of 250-300 bp following the previously described procedure [45 (link), 46 (link)]. The sequences of primers used for qPCR were given in Supplementary Table 5 and their location is depicted in Figure 4B. Although the amplicons are not tiled, the average size of chromatin fragments ensured that CTCF is absent from the entire region under consideration. Epigenetic modifications of histones were studied at nucleosomal level by Nuc-ChIP [31 (link), 32 (link)]. The following antibodies were used: anti-H3K9ac (Abcam, ab-4441); anti-H3K9me3 (Abcam, ab-8898); anti-H3K27ac (Abcam, ab-4729); anti-H3K27me3 (Millipore, 07-449); anti-H3K4me3 (Abcam, ab-8580); anti-H3K36me3 (Abcam, ab-9050); anti-H3K20m (Abcam, ab-9051); anti-β-actin (Abcam, ab-8227).

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.

The regular chromatin preparation was performed as described^{50 (link)}. In brief, cells were cross-linked with 1% formaldehyde solution for 10 min and quenched with 0.125 M glycine. For H3R8me2a ChIP, the cell pellets were resuspended in cold CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES, pH 6.8) containing Triton X-100 (0.5%) and EGTA (1 mM) before 10 min of fixation in 0.5% Formaldehyde solution. After being quenched by 0.125 M Glycine, cells were spun down, washed, resuspended, lysed, and ultra-sonicated for 25 min with 30 s ultra-sonication at 30 s intervals (Bioruptor pico, Diagenode, Belgium). The resulting fragmented chromatin extract was precleared with Protein A/G beads (ThermoFisher, Beijing, China) and then incubated overnight with antibodies: anti-H3K4me1, anti-H3K4me3, anti-3K27me3, anti-H3K9me3 (Cell Signaling Technology), anti-H3K27ac (Abcam), anti-H3R8me2a (Novus Biologicals), and Normal anti-rabbit IgG (Cell Signaling Technology) as controls. After stringent washes, elution, and reverse cross-linking, DNA was purified using PCR purification kits (QIAGEN, Hilden, Germany). The primers for ChIP-qPCR analyses at the promoters and enhancers are listed in Supplementary Table 2.