Anti h3k27ac antibody

To investigate the roles of SRSF2 in the status of histone modification near the TSS of genes, ChIP assay was conducted according to Dahl’s protocol [56 (link)]. In brief, 1 × 10⁶ Jurkate E6 cells transfected with SRSF2 siRNA or control siRNAs were fixed with 1% formaldehyde and sonicated on ice for 10x10 s, with 20 s pauses between each 10 s session and 30% power using a probe sonicator (Sonics & Materials, Inc.). After centrifugation, the supernatants were incubated with anti-H3K4Me3 antibody (Abcam, ab8580), anti-H3K27Me3 antibody (Abcam, ab6002), anti-H3K27Ac antibody (Abcam, ab4729), or anti-STAT3 Y705 antibody (Abcam, ab76315). Chromatin DNA was purified by Dynabeads protein G (Invitrogen, 10004D) and subjected to real-time PCR. The region-specific primers used are listed in Table S1.

For chromatin immunoprecipitation (ChIP), cells were harvested and fixed after stimulation. Samples were lysed and sonicated using a Branson 1200 Ultrasonic Cleaner to obtain chromatin fragments of optimal size (300-800 bp). Sheared chromatin was incubated using 2 μL of anti-H3K27ac antibody (Abcam, ab4729) or 2 μL of anti-IgG antibody (Millipore, 12-370), and immunoprecipitated with Dynabeads A (Invitrogen, 10001D) and Dynabeads G (Invitrogen, 10003D) magnetic beads. ChIP samples were de-cross-linked with proteinase K (Thermo Fisher Scientific) at 65°C for 4 h. Then, DNA was purified with the DNA Clean Kit & Concentrator TM-5 (Zymo Research). Quantitative analysis of the purified ChIP and Input DNAs was performed by using FastStart Universal SYBR Green Master (Roche) by real time qPCR.

The parental and BRD9‐KO COAD cells were collected and subjected to ChIP assay. These cells were harvested and sonicated by bioruptor (Diagenode). By using chromatin immunoprecipitation (ChIP) assay kit (Upstate, cat no. 17‐371), the supernatant of sonicated cells was co‐immunoprecipitated with the anti‐BRD9 (Cell signaling Technology, CST#58906), or the anti‐H3K27ac antibody (Abcam, ab4729), or the IgG antibody as the negative control. The enrichment of target gene fragment in DNA precipitate was analyzed by quantitative real‐time PCR. The fold enrichments of target genes between ChIP‐DNA and input‐DNA were determined by ΔCt.

Anti-BRD4 and anti-H3K27ac Chip-qPCR of POU5F1B superenhancer was performed, respectively. The forward and reverse primers are listed in supplementary table 2. Chromatin immunoprecipitation (ChIP) assay was performed according to the protocol developed by Upstate Biotechnology. Briefly, chromatin lysates were prepared from hepatocytes after crosslinking with 1% formaldehyde. The samples were precleared with Protein-G agarose beads and immunoprecipitated using anti-H3K27ac antibody (Abcam, #cat: ab4729), anti-BRD4 antibody (Abcam, #cat: ab128874), or control anti-IgG (Abcam, #cat: ab172730) in the presence of BSA. Beads were extensively washed before reverse crosslinking. DNA was purified using a PCR Purification Kit and subsequently analyzed by qPCR.

The ChIP-seq and ChIP-qPCR assays were carried out as previously described [32 (link), 33 (link)] with modifications. Briefly, H1299 cells were fixed with 1% formaldehyde, lysed in FA lysis buffer with protease inhibitor Complete cocktail. Chromatin was sonicated with a sonifier (Qsonica) followed by immunoprecipitated with anti-H3K27ac antibody (Abcam, ab4729). For ChIP-seq assays, 1 × 10⁷ cells and 3 μg antibodies were used, and isolated genomic DNAs were subjected to library construction and high-throughput DNA sequencing by the Genomic Research Center of National Yang Ming Chiao Tung University (Taiwan). Raw reads were mapped to hg19 human genome assembly, H3K27ac peaks were called by MACS software with default parameters and input DNA as control. Super-enhancer (SE) analysis was performed using the ROSE program [34 (link)] with all H3K27ac peaks identified in WT and/or USP7 KO H1299 cells. For ChIP-qPCR assays, 3 × 10⁶ cells and 1 μg antibodies were used. Quantitative PCRs were carried out with QuantiNova Probe PCR kit (Qiagen) and the ΔΔCt method were used to determine the enrichment of indicated factors. The antibodies used for ChIP-qPCR were anti-SMAD3 (CST and C67H9), anti-SMAD4 (CST and D3R4N), and anti-USP7(Bethyl and A300-033A). All ChIP-qPCR primers are listed in Supplementary Table S1.

Anti-TRIM33 antibody (A301-060A; Bethyl Laboratories, Montgomery, TX), Anti-PU.1 antibody (#2266; Cell Signaling, Beverly, MA or sc-352; Santa Cruz, Dallas, TX), Anti-Bim antibody (#2819; Cell Signaling), Anti-H3K27ac antibody (ab4729; Abcam, Cambridge, MA), Anti-H3K4me3 antibody (07-473; Millipore), Anti-ß-actin HRP antibody (#A3854; Sigma, Ronkonkoma, NY), APC anti-mouse B220 (#103212; BioLegend, San Diego, CA), APC anti-mouse CD-19, APC anti-mouse Mac-1/Cd11b (#101211; BioLegend), APC anti-mouse Ly-6G/Gr-1 (#17-5931; eBioscience, San Diego, CA), APC anti-mouse TER-119 (#116212; BioLegend), APC anti-mouse CD-3 (#100209; BioLegend).