Anti h3k27ac ab4729

CUT&Tag libraries were prepared using the Hyperactive in situ ChIP Library Prep Kit for Illumina (TD901, Vazyme Biotech, Nanjing, China). Briefly, cells were counted, and 100,000 cells were used for each assay. These cells were bound to the conA beads and incubated with anti-H3K27ac (ab4729, Abcam, Cambridge, UK) or anti-Nipbl antibody (A301-779A, Bethyl Laboratories, Montgomery, TX, USA) for 2 h at room temperature. After washing, cells were incubated with a secondary antibody (Goat Anti-Rabbit IgG, Sangon, Shanghai, China), and diluted (1:100) in the DIG Wash buffer for 1 h on a shaker with slow rotation at room temperature. The Hyperactive pG-Tn5 Transposase was then added, and cells were incubated for 1 h. After washing three times with the Dig-300 buffer, these cells were resuspended in the tagmentation buffer and incubated for 1 h at 37 °C. Chromatin was purified using a standard phenol/chloroform/alcohol extraction method. Fragmented DNA was then amplified by PCR and then purified by magnetic beads. The final libraries were sequenced on an Illumina platform.

The lentiviral shRNA plasmids pLKO.1 targeting mouse Brd4 (clone IDs TRCN0000311976, TRCN0000088480, and TRCN0000088481) were purchased from Sigma. Anti-RbBP5 (A300–109A), anti-Brd4 (A301–985A100), and anti-MED1 (A300–793A) were from Bethyl Laboratories. Anti-C/EBPα (sc-61X), anti-C/EBPβ (sc-150X), anti-PPARγ (sc-7196X), and anti-p300 (sc-585X) were from Santa Cruz Biotechnology. Anti-H3K4me1 (ab8895), anti-H3K4me2 (ab7766), and anti-H3K27ac (ab4729) were from Abcam. Anti-Pol II (17-672) was from Millipore. For western blot analysis, all antibodies were diluted to 1 μg ml⁻¹. Uncropped blots are available in the Supplementary Figure 7.

ChIP assays were performed as described [71 (link)] using anti-CHD8 (A301-224A) from Bethyl Laboratories or home-made rabbit anti-CHD8 [16 (link)], anti-RNAPII (N-20) (sc-899), anti-PR (H-190) (sc-7208), anti-BAF155 (R-18) (sc-9746) and anti-FOXA1 (H-120) (sc-22841) from Santa Cruz Biotechnology, and anti-H3K27Ac (ab4729) from Abcam. Chromatin was sonicated to an average fragment size of 400 to 500 bp using the Diagenode Bioruptor. Rabbit IgG (Sigma) was used as a control for non-specific interactions. Input was prepared with 10% of the chromatin material used for immunoprecipitation. Input material was diluted 1:10 before PCR amplification. Quantification of immunoprecipitated DNA was performed by real-time PCR (qPCR) with the Applied Biosystems 7500 FAST real-time PCR system, using Applied Biosystems Power SYBR green master mix. Sample quantifications by qPCR were performed in triplicate. Sequences of all oligonucleotides are available upon request. Data are the average of at least three independent experiments.