We used two ovaries from different animals for each biological replicate; we used two replicates for control and treated groups. Chromatin immunoprecipitation in adult tissue was performed as previously described [2 (link)], except for small modifications. Specifically, chromatin was not dialyzed after sonication but instead diluted 6 times with ChIP-dilution buffer. We performed ChIP using 1 µg of H3K4me3 (Merck Millipore, 07-473). Immunoprecipitated DNA was purified with Qiagen MinElute kit, and 10 µg of DNA was used for library construction. Library was prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645) and submitted to Genomic platform to perform massively parallel 50-bp sequencing in single-end mode.
H3k4me3
Manufactured by Merck Group
Sourced in United States, United Kingdom, Belgium
H3K4me3 is a histone modification marker that indicates the presence of trimethylation of lysine 4 on histone H3. This histone modification is associated with active transcription and is commonly used as a marker for identifying actively transcribed genomic regions.
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Lab products found in correlation
H3k27me3, by Merck Group (66 mentions)
H3k27ac, by Abcam (23 mentions)
H3k9ac, by Merck Group (20 mentions)
H3k4me2, by Merck Group (19 mentions)
H3k9me3, by Abcam (19 mentions)
H3k27ac, by Merck Group (16 mentions)
H3k4me1, by Merck Group (14 mentions)
H3k9me3, by Merck Group (14 mentions)
H3k36me3, by Abcam (13 mentions)
H3k9me2, by Abcam (13 mentions)
H3k4me1, by Abcam (12 mentions)
Dynabead, by Thermo Fisher Scientific (9 mentions)
H3k9me2, by Merck Group (9 mentions)
Bioruptor, by Diagenode (7 mentions)
H3k27me3, by Abcam (6 mentions)
H3k27me2, by Merck Group (6 mentions)
H3k36me2, by Abcam (6 mentions)
Hiseq 2500, by Illumina (6 mentions)
Ab9050, by Abcam (5 mentions)
Flag tag (flag), by Merck Group (5 mentions)
186 protocols using h3k4me3
1
ChIP-seq on Adult Tissue
2
Chromatin Immunoprecipitation (ChIP) Assay
3
Chromatin Immunoprecipitation Protocol
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ChIP was performed as described previously43 (link). Briefly, fixation of cells was done with formaldehyde and DNA was subsequently sheared by sonification with a Bioruptor (high, 15 cycles: 30′′on, 30′′off) (Diagenode, Liège, Belgium). For immunoprecipitation, Dynabeads (Invitrogen) were incubated 15 min with 5 μg of specific antibodies H3K4me3 (07–473; Merck-Millipore, The Netherlands), H3K79me2 (ab3594; Abcam), H3K79me3 (ab2621; Abcam), normal rabbit IgG (ab46540; Abcam), normal mouse IgG (12–371; Merck-Millipore), anti-FLAG (f1804; Sigma-Aldrich) and anti-HA (101P-200; Covance, The Netherlands)) in 0.02% PBS–Tween-20, then unbound antibodies were washed off, and diluted sheared chromatin was added to the complex of magnetic Dynabeads-antibody (rotating overnight at 4 °C). After washing and elution with 2% SDS and 50 mmol l−1 NaHCO3, samples were treated with RNase and Proteinase K (Roche). DNA was purified using the Qiaquick DNA spin columns (Qiagen, Venlo, Netherlands) according to the protocol. Subsequently, RT–PCR was performed with AbsoluteQPCR SYBRGreenROXMix (Thermo Scientific) using specific primers (Supplementary Table 5 ). The % input was expressed as AE (Ct input−Ct ChIP) × Fd × 100%, where Fd is a dilution compensatory factor and AE represents the primer efficiency.
4
Western Blot Characterization of Histone Modifications
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Cells were washed once with cold PBS and lysed by RIPA lysis buffer and incubated with 4 × SDS loading buffer at 95 °C for 10 min. Whole cell proteins were used for electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked with Tris buffered saline containing 0.075% Tween-20 and 5% non-fat milk. Then the membranes were incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with secondary antibodies for 1 h at room temperature.
Antibodies used in this study included H3 (Cell Signaling Technology, 9715; dilution 1:1000), H3K27me3 (Cell Signaling Technology, 9733; dilution 1:1000), H3K27me2 (Cell Signaling Technology, 9728; dilution 1:1000), H3K27me1 (Merck Millipore, 07-448; dilution 1:1000), H3K27ac (Cell Signaling Technology, 8173; dilution 1:1000), H3K4me3 (Merck Millipore, 04-745; dilution 1:1000), H3K36me3 (Cell Signaling Technology, 4909; dilution 1:1000), H3K9me3 (Active Motif, 39161; dilution 1:1000), H3K79me2 (Abcam, ab3594; dilution 1:1000), p16 (Santa Cruz, sc-377412; dilution 1:200), β-actin (Cell Signaling Technology, 3700, dilution 1:1000) (Supplementary Table1 ).
Antibodies used in this study included H3 (Cell Signaling Technology, 9715; dilution 1:1000), H3K27me3 (Cell Signaling Technology, 9733; dilution 1:1000), H3K27me2 (Cell Signaling Technology, 9728; dilution 1:1000), H3K27me1 (Merck Millipore, 07-448; dilution 1:1000), H3K27ac (Cell Signaling Technology, 8173; dilution 1:1000), H3K4me3 (Merck Millipore, 04-745; dilution 1:1000), H3K36me3 (Cell Signaling Technology, 4909; dilution 1:1000), H3K9me3 (Active Motif, 39161; dilution 1:1000), H3K79me2 (Abcam, ab3594; dilution 1:1000), p16 (Santa Cruz, sc-377412; dilution 1:200), β-actin (Cell Signaling Technology, 3700, dilution 1:1000) (Supplementary Table
5
Antibody Generation and Characterization
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The antibody against mouse C17orf96 was generated by immunizing rabbits (Covance, Princeton, NJ, USA) with a peptide corresponding to the N terminus of mouse C17orf96 (LKPRRGTPEFSPLC). Sera were obtained and the antibodies were positively selected using a purified GST-PLKPRRGTPEFSPLCL fusion protein and negatively selected using bacterial crude cell extract, each coupled to CNBr-activated Sepharose beads. Other used antibodies were Suz12 (Cell Signaling, no. D39F6 (ChIP); Santa Cruz, Dallas, TX, USA, sc-46264/sc-271325 (Western)), p300 (Santa Cruz, sc-585), Tubulin (Sigma-Aldrich, T9026), actin (Abcam, Cambridge, MA, USA, ab3280), histone H3 (Abcam, ab1791), H3K27me3 (EMD Millipore, Billerica, MA, USA, no. 07-449), H3K4me3 (EMD Millipore, no. 04-745), Flag M2 (Sigma-Aldrich, F1804), Flag M2 beads (Sigma-Aldrich, A2220) and HA.11 (MMS-101P, Covance).
6
ChIP-qPCR for Histone Marks
7
Quantitative Immunohistochemistry of Histone Modifications
8
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were performed using the EZ-Magna ChIP assay kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, cells were washed with PBS and cross-linked with 1% formaldehyde for 10 min. Chromatin was sonicated on ice to generate chromatin fragments of 200–1000 bp, and then immunoprecipitated with each antibody. Antibodies for acetylated histone H3 (AcH3, Merck Millipore), tri-methylated histone H3K4 (H3K4me3, Merck Millipore), and isotype IgG (negative control, Cell Signaling Technology) were used in the immunoprecipitations. The immunoprecipitated materials were then washed extensively, and cross-linking was reversed. DNA from the eluted chromatin was purified by phenol extraction and ethanol precipitation. Input control DNA or immunoprecipitated DNA was determined by quantitative real-time PCR. Standard curves were constructed using a pool of input samples, and each ChIP sample was normalized to its respective input. We designed primers to separately amplify five regions in the FABP4 promoter region. The primer pairs used for ChIP assays are shown in Supplementary Table S1 .
9
CUT&Tag: Profiling Chromatin Modifications
10
Quantifying Histone Modifications by ChIP-seq
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Chromatin immunoprecipitation (ChIP) was performed as described previously40 (link). Cells were transfected with siRNAs as described above and incubated for 72 h. Aliquots of cells (1 × 106 (link)) were then fixed and crosslinked, after which samples were immunoprecipitated for 12 h at 4 °C using a rabbit anti-acetylated histone H3 lysine 27 (H3K27ac) pAb (#39,133; Active Motif, Carlsbad, CA, USA), a rabbit anti-trimethylated histone H3 lysine 4 (H3K4me3) mAb (#04–745; Merck, Darmstadt, Germany), or normal mouse IgG (#140–09,511, Fujifilm, Tokyo, Japan), which served as a control. For ChIP-seq analysis, samples were sequenced using an Ion Proton System (Thermo Fisher Scientific). Sequencing data were mapped to the human genome hg19 using bowtie2, after which peaks were called using MACS2 and were annotated using HOMER v4.11. ChIP-seq results were visualized using Microsoft Excel, Java TreeView and Integrative Genomics Viewer. ChIP-qPCR was carried out using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). The sequences of the PCR primers are listed in Supplementary Table S2 . The Gene Expression Omnibus accession number for the microarray data is GSE178613.
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