CHL-1 cells were treated for 4 h with 500 nM BAY 1238097 or OTX-015 prior to cross-linking and harvest. One 15 cm cell culture dish with 80% confluent cells was used per immunoprecipitation. ChIP DNA preparation for sequencing was carried out as described previously38 (link) with the following changes: cross-linked chromatin was sheared to 200–800 base pair fragments using sonication (30 s on/30 s off, high power, 2 × 7.5 min with a water bath change between the two cycles) in a Bioruptor sonicator UCD-200 (Diagenode, Liège, Belgium). About 1.5 μg antibodies (anti-BRD4 (A301-985A100, Bethyl Labs, Montgomery, TX, USA); anti-H3K27ac (Diagenode, C15410174)) were used per immunoprecipitation. DNA was purified using a PCR purification kit (Qiagen), followed by library preparation and genome-wide sequencing. In brief, samples were analyzed on a 2100 bioanalyzer using the DNA high sensitivity assay. Concentration was determined using a High Sensitivity DNA Kit. End repair and phosphorylation, and ligation of TrueSeq index adapter and amplification before subjection to sequencing (HiSeq Sequencing System) were done according to the TrueSeq ChIP Sample Preparation Guide (Illumina, Munich, Germany). Sequencing data are available in the GEO database with the accession number GSE95585.
Anti h3k27ac
Manufactured by Diagenode
Sourced in United States, Germany
Anti-H3K27ac is a lab equipment product from Diagenode. It is a specific antibody that binds to the acetylated form of histone H3 at lysine 27 (H3K27ac). This antibody can be used in various applications, such as chromatin immunoprecipitation (ChIP) and Western blotting, to detect and analyze the presence and distribution of the H3K27ac histone modification.
Automatically generated - may contain errors
Lab products found in correlation
Anti h3k4me1, by Diagenode (3 mentions)
Anti h3k4me3, by Diagenode (2 mentions)
Anti h3k27me3, by Diagenode (2 mentions)
C15410196, by Diagenode (2 mentions)
Hpa003259, by Merck Group (2 mentions)
Bioruptor ucd 200 sonicator, by Diagenode (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Anti brd4 a301 985a100, by Fortis Life Sciences (1 mentions)
Anti h3k9me3, by Diagenode (1 mentions)
Anti ezh2 d2c9, by Cell Signaling Technology (1 mentions)
Anti h2ak119ub d27c4, by Cell Signaling Technology (1 mentions)
Anti cbx8, by Diagenode (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Superscript vilo, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Anti p63 4a4, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti h3k27ac
1
ChIP-seq Analysis of BRD4 and H3K27ac
2
Histone Modifications and Transcription Factors ChIP-seq
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The following antibodies targeting histone modifications were used for ChIP-seq (1 μg per ChIP for cell numbers below 100,000, 2 μg per ChIP for cell total cell numbers per ChIP below 250,000): anti-H3K27ac (C15410196, lot A1723–041D), anti-H3K4me3 (C15410003, lot A5051–001P), anti-H3K4me1 (C15410194, lot A1863–001D), anti-H3K36me3 (C15410192, lot A1847–001P), anti-H3K9me3 (C15410193, A1671–001P), anti-H3K27me3 (C15410195, lot A1811–001P), all from Diagenode. Transcription factors antibodies: CTCF (3 μg/ChIP, Abcam, ab70303), p300 (10 μg/ChIP, Santa Cruz, sc-585).
3
ChIP Analysis Protocol for Epigenetic Markers
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ChIP analysis was essentially performed as described previously (Frank et al., 2001 (link)). ChIP reactions were performed using the following antibodies: anti-GFP (ab290, Abcam), anti-CBX8 (C15410333, Diagenode), anti-EZH2 (D2C9, Cell Signaling), anti-H2AK119ub (D27C4, Cell Signaling), anti-H3K27me3 (C15410195, Diagenode), anti-H3K27Ac (C15410196, Diagenode), anti-H3K4me1 (C15410194, Diagenode), and anti-H3K4me3 (C15410003, Diagenode). ChIP efficiencies were assessed using qPCR. Primer sequences can be found in Supplementary file 6 .
4
RNA and Chromatin Immunoprecipitation Analysis
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RNA was extracted in TRIzol reagent (Invitrogen) and treated with DNase I (Roche). Complementary DNA (cDNA) synthesis was obtained using SuperScript Vilo (Invitrogen). Expression of target genes was normalized for the β-actin gene expression. ChIP analysis was performed as previously described (43 (link)) using rabbit polyclonal antibody anti-p63 (H137, Santa Cruz Biotechnology), anti-histone H3K4me3 (Upstate 07–473), anti-H3K27Ac (Diagenode), anti-Cebpa (14AA, Santa Cruz Biotechnology), anti-Cebpb (C-19, Santa Cruz Biotechnology), anti- Pou3f1 (kindly provided by Dr. Michael Wegner, Universität Erlangen-Nürnberg, Germany) (55 (link)) or rabbit anti-mouse IgG (Santa Cruz Biotechnology). Real-time RT-PCR was performed using the SYBR Green PCR master mix (Applied Biosystems) in an ABI PRISM 7500 (Applied Biosystems). Oligonucleotide sequences are given in the Supplementary Data. For immunoblotting, cells were lysed in sample buffer and immunoblotted as previously described (43 (link)). The following primary antibodies were used: anti-p63 (4A4, Santa Cruz Biotechnology), anti-β-actin (Santa Cruz Biotechnology). For immunoblotting, secondary antibodies were sheep anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare) and visualized using Enhanced chemiluminescence (ECL) western blotting system (GE Healthcare).
5
Chromatin Immunoprecipitation Sequencing
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ChIP-Seq was performed as previously described [26 (link)], using anti-H3K27ac (C15410196, Diagenode) or anti-MITF (Sigma, HPA003259). Details of ChIP-seq and data analysis are described in the supplemental information . The human tissue samples used in this study were collected on protocol 2007P000699 “Culture of LAM, AMLs, and other TSC Lesions” approved by the Partners Human Research Committee and informed consent was obtained from all subjects.
Additional methodologic details for metabolomic profiling, whole transcriptome RNA sequencing and immunohistochemistry are provided insupplemental information .
Additional methodologic details for metabolomic profiling, whole transcriptome RNA sequencing and immunohistochemistry are provided in
6
ChIP-seq of Histone Modifications in Mouse Embryonic Gonads
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Gonads from E13.5 mouse embryos were fixed using 1% formaldehyde and subsequently snap-frozen and stored at −80°C. Chromatin immunoprecipitations were performed using the iDeal ChIP-seq Kit for Histones (Diagenode, C01010051) according to the manufacturer's instructions. Briefly, whole fixed gonads were lysed and subsequently sonicated using a Bioruptor (45 cycles, 30 s on, 30 s off, at high power) in the provided buffers. Sheared chromatin (5 µg per immunoprecipitation) was then used with 1 µg of the following specific histone antibodies: anti-H3K4me3 (Millipore, 07-473), anti-H3K4me1 (Diagenode, C15410037) and anti-H3K27ac (Diagenode, C15410174). The samples were sequenced using Illumina HiSeq technology according to standard procedures. Mapping was performed with the STAR v2.6.1d software41 using settings to enforce unspliced read mapping (--alignEndsType EndToEnd --alignIntronMax 1 --outFilterMatchNminOverLread 0.94). Finally, de-duplication was performed via bamUtil (version 1.0.14; option –rmDups, https://github.com/statgen/bamUtil/releases ). Previous published ChIPseq data from mole developing gonads (Real et al., 2020 (link)) were used to call putative enhancer regions.
7
Chromatin Immunoprecipitation Sequencing
8
ChIP Assay for Chromatin Profiling
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ChIP was performed using MAGnify Chromatin Immunoprecipitation System (Invitrogen) following the manufacturer's protocol. Chromatin was sonicated at 25% amplitude for 15 min (20″ sonication/30″ pause) using SONOPULS UW3100 (BANDELIN electronics). End‐point PCRs were performed using GoTaq G2 Flexi DNA Polymerase (Promega) according to the manufacturer's protocol. Real‐time PCR (Fig 5 C) was performed using GoTaq qPCR Master Mix (Promega); the primers used are listed in Appendix Table S5 . The antibodies used were as follows: anti‐ACTL6A (Cell Signaling, 76682S); anti‐SMARCC2 (Cell Signaling, D809V, 12760S); and anti‐H3K27ac (Diagenode, C15410174).
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