Sc 585x

Chromatin was extracted from naive CD4⁺Foxp3⁻ T cells (1 × 10⁶ cells) polarized for 24–72 h under various conditions after fixation with formaldehyde. Anti-Histone H3ac (39139, 7 μl), anti-Histone H4ac (39243, 6 μl), anti-Histone H3 (trimethyl K9) (61013, 7 μl), anti-Histone H3 (dimethyl K27) antibody (39245, 5 μl; all from Active Motif), anti-HDAC1 (ab7028, 5 μg), anti-Smad3 (ab28379, 5 μg), anti-Histone H3 (trimethyl K27) (ab6002, 5 μl), anti-Histone H3 (acetyl K27) (ab4729, 3 μl), anti-Histone H3 (acetyl K9) (ab4441, 4 μl; all from Abcam), anti-dimethyl-Histone H3 (diMe-Lys9) (D5567, 5 μl; Sigma- Aldrich), anti-p50 (sc-1192X, 5 μg), anti-p300 (sc-585X, 5 μg), anti-STAT6 (SC-981X, 5 μg), control mouse IgG (sc-2025, 5 μg; all from Santa Cruz Technology) and purified goat IgG (02–6202, 5 μg; Life Technologies) were used for the immunoprecipitation of chromatin with an EZ ChIP kit (17–371; EMD Millipore) according to the manufacturer's instructions. The precipitated DNA was measured by real-time PCR using SsoAdvanced Universal SYBR Green Supermix (172–5275; Bio-Red). Real-time PCR primer sequences are listed in Supplementary Table 2. Data are presented as relative binding based on normalization to input DNA50 (link).

ES cells were cultured in feeder-free conditions using N2B27 + 2i media supplemented with 2000U mL⁻¹ of ESGRO (Millipore) on a gelatinized plate. KH2 ES cells^{72 (link)} with epitope-tagged Hoxb1 (3XFLAG-MYC) were used for Hoxb1 ChIP using anti-flag antibody (F1804-Sigma). Unmodified KH2 cell lines were used for ChIP experiments for Pbx (SC-888; Santa Cruz), Meis (SC-25412; Santa Cruz), Prep1 (ab55603; Abcam), Prep2 (sc-292315X; Santa Cruz) and EP300 (Sc-585X; Santa Cruz). Cells were differentiated to neuroectoderm in differentiation media containing DMEM + 10% (vol/vol) Serum + NEAA + 3 µM RA for a requisite length of time. Cells were harvested at 80–90% confluency.

ChIP was performed according to standard protocol [50 (link)] with minor modifications. Paraformaldehyde (1 %) cross-linking was carried out for 10 min followed by the chromatin preparation as described earlier [7 (link)]. Nuclei were re-suspended in ChIP-incubation buffer at a concentration of 20 × 10⁶ cells/mL and sheared (seven cycles with each cycle containing 10 s power on and 10 s interval) using Bioruptor®Plus (B01020001, Diagenode, Liege, Belgium). Sonicated chromatin equivalent of 4 × 10⁶ cells was incubated with relevant antibody overnight at 4 °C. Antibodies against P300 (sc-585x, Santa Cruz Biotechnology, Inc., Dallas, Texas, United States), POLII (MMS-126R-500, Covance, Inc., Princeton, New Jersey, United States), H3K27ac (C15410196, Diagenode), H3K4me1 (C15410194, Diagenode), and H3K4me3 (C15410003, Diagenode) were used. ChIP-seq sample preparation and sequencing was performed according to manufacturer’s instructions (Illumina, San Diego, California, United States) and essentially as described [6 (link), 9 (link), 51 (link)] (http://www.blueprint-epigenome.eu).