Isolated monocyte-enriched suspensions were fixed with 1% formaldehyde (Sigma). Fixed cell suspensions were sonicated using a Bioruptor Pico (Diagenode) for seven cycles (30 s on; 30 s off). For each Chromatin immuno precipitation (ChIP), chromatin of 0.5×106 cells was incubated with 254 μL dilution buffer, 12 μL protease inhibitor cocktail (25×), and 1 µg of H3K4me3 antibody (Cell Signaling Technology, Danvers USA) and incubated overnight at 4C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4C. Beads were washed with 500 mL buffer for 5 min at 4C with five rounds of washes. After washing, chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 µL 5M NaCl, 2 µL proteinase K were added and samples were incubated on a shaking heat-block for 4 hours at 1000 rpm, 65C. This elution procedure was also repeated for chromatin input samples, where 0.5×106 cells were used for input as well. As final step, the DNA was isolated using QIAGEN MinElute PCR purification Kit and eluted in 20 µL elution buffer.
H3k4me3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The H3K4me3 antibody is a laboratory reagent used to detect and quantify the presence of histone H3 protein that is trimethylated at lysine 4. This modification is associated with active gene transcription. The antibody can be used in various applications, such as chromatin immunoprecipitation (ChIP), Western blotting, and immunofluorescence microscopy, to study epigenetic regulation of gene expression.
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Lab products found in correlation
Bioruptor pico, by Diagenode (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Genelute pcr clean up kit, by Merck Group (1 mentions)
Simplechip plus enzymatic chromatin ip kit magnetic beads, by Cell Signaling Technology (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
H3k27me3 antibody, by Cell Signaling Technology (1 mentions)
H3k9me3 antibody, by Active Motif (1 mentions)
H3k4me1 antibody, by Active Motif (1 mentions)
H3k27ac antibody, by Active Motif (1 mentions)
Pierce agarose chip kit, by Thermo Fisher Scientific (1 mentions)
H3k9ac antibody, by Cell Signaling Technology (1 mentions)
H3k4 me2 antibody, by Cell Signaling Technology (1 mentions)
H3k9me2 antibody, by Cell Signaling Technology (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
8 protocols using h3k4me3 antibody
1
ChIP-seq protocol for H3K4me3 analysis
2
Genome-wide Chromatin Profiling via ULI-NChIP
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ChIP-seq library was prepared according to the ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) procedure65 (link). Briefly, 50,000 sorted cells were used per reaction. The sorted cells were washed two times in 0.5% BSA/PBS solution. The nuclei were collected and subjected to fragmentation reaction in a preheated metal bath at 37 °C for 5 min. After immunoprecipitation reaction with desired antibody, DNA fragments were extracted were purified using Phenol-Chloroform. One microgram of either H3K27me3 antibody (pAb-069-050, Diagnode) or H3K4me3 antibody (9727, Cell signaling Technology) was used for each reaction. Antibodies used were described in supplementary Table 1 . Sequencing libraries were prepared according to KAPA Hyper Prep Kit with minor modifications. All the generated DNA libraries were sequenced on the HiSeq X Ten platform.
3
Chromatin Immunoprecipitation (ChIP) from Tissue
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ChIP using tissue was performed according to the manufacturer's instructions (Abcam). Briefly, ~20 mg frozen EPN tissue was minced into 1–3 mm3 pieces, thawed on ice, washed with ice cold PBS with proteinase inhibitor cocktail prior to fixation in 1% formaldehyde in PBS at room temperature for 10 min and quenched with 0.125 M glycine for 5 min. Tissue was collected and resuspended in 500 μl FA-lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL pepstatin) and disrupted while on ice, with a Wheaton Dounce Tissue Grinder (VWR International, 62,400–595). The tissue suspension was transferred into a 5 ml conical tube, sonicated and processed according to manufacturer's protocol using rabbit polyclonal H3K4me3 antibody (Cell Signaling Technology, Cat# 9727) and IgG from rabbit serum (Cell Signaling Technology, Cat# 2729) to collect immunoprecipitated DNA fragments. All DNA fragments were cleaned-up with GenElute PCR clean-Up Kit (Sigma Aldrich Co., St. Louis, MO, USA) prior to PCR analysis. ChIP using cells was performed with the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic beads) (Cell Signaling Technology, Cat#9005) following manufacturer's instructions. Human ERBB2 and Cyclin D1 ChIP primers (Supplementary Table 3) were used following real-time PCR analysis.
4
ChIP Assay for H3K4me3 Markers
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ChIPs were performed using the ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY, USA) and the H3K4me3 antibody (9751) from Cell Signaling Technology. Primers used for PCR detection are listed as follows: 5′-CCCGCCCCAGCTGTGTCATTTT-3′ and 5′-AATGGTGCCCATCCACGTGG-3′ for E-cadherin (−80 to +88); 5′-CCAAAGTGCTGGTATTCCGCTGTAAG-3′ and 5′-GTGTGCTCCCAGAGTCGGGTTTGC-3′ for N-cadherin (−5,112 to −4,961), 5′-GGTGTGGTTTCATGGGGGGAGG-3′ and 5′-CCCTAAGTTTTTAATAACTCGCTAAAG-3′ for vimentin (−116 to +91).
5
Chromatin Profiling of Pluripotent Cells
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For ULI-NChIP-seq, 10 000 or 40 000 cells were used per reaction. The ULI-NChIP procedure was performed as previously described (17 (link),18 ). Briefly, 2 μg of H3K27ac antibody (39133, Active Motif), H3K4me1 antibody (39297, Active Motif), H3K4me3 antibody (9727, Cell Signaling Technology), H3K27me3 antibody (9733, Cell Signaling Technology) or H3K9me3 antibody (39161, Active Motif) was used for each immunoprecipitation reaction. The sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, New England Biolabs) following the manufacturer's instructions. The CUT&Tag assays for CTCF were performed using 50 000 sorted ESCs and 2CLCs as previously described (19 (link)). Two or three replicates were performed. Barcoded libraries were pooled and sequenced on an Illumina HiSeq X Ten platform in the paired-end mode.
6
Chromatin Immunoprecipitation Assay Protocol
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ChIP assays were performed using Pierce Agarose ChIP Kit (#26156, Thermo) according to the manufacturer’s protocol. In brief, after transfection for 72 h, 1% formaldehyde was added to the medium and porcine GCs were cross-linked for 10 min, and then under 6U of micrococcal nuclease had been added in a 37 °C water bath for 15 min. H3K4me2 antibody (#9725 S), H3K4me3 antibody (#9751 S), H3K9me2 antibody (#4658 S) and H3K9ac antibody (#9649 S) were all obtained from Cell Signaling Technology (USA), and were used to pull down the immunoprecipitated complex. After pull down, the fragments of interest were detected by PCR and the amplified products were analyzed by 3% agarose gel electrophoresis, and the enrichment was normalized to the IgG group. IgG antibody (#2985 S, Cell Signaling Technology) was used as a negative control and unprocessed DNA served as the input control. The primers used for the ChIP assay are listed in Table S5 .
7
Chromatin Immunoprecipitation Sequencing
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Forty milliliters of yeast culture were combined with 1.11 mL of 37% formaldehyde (1% final concentration) in a 50 mL Falcon tube. The lid was tightened to prevent leakage, and the tube was placed in a silent mixer at room temperature for 25 min. Afterward, 2.74 mL of 2 mol L -1 glycine was added and mixed at room temperature for 10 min to stop crosslink reactions. The mixture was washed three times with 20 mL of cold (4°C) PBS and resuspended with 3 mL of PBS that contained protease inhibitor. A standard ChIP-seq protocol (Kim and Dekker, 2018) (link) was carried out using an H3K4me3 antibody (Cell Signaling Technology, USA) and then sequenced on the Illumina NovaSeq6000, using the PE150 mode. The ChIP-seq analysis was similar to the ATAC-seq analysis. The ChIPed and input sample pairs were mapped to the reference genome using Bowtie2, and enriched peaks were called using MACS2. The mapped bam files were finally converted to Bigwig format via bam-Coverage using RPKM normalization for visualization.
8
H3K4me3 ChIP-seq protocol
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Isolated monocyte-enriched suspensions were fixed with 1% formaldehyde (Sigma). Fixed cell suspensions were sonicated using a Bioruptor Pico (Diagenode) for 7 cycles (30s on; 30s off).
For each ChIP, chromatin of 0.5x10^6 cells was incubated with 254 uL dilution buffer, 12uL protease inhibitor cocktail (25x), and 1µg of H3K4me3 antibody (Cell Signaling Technology, Danvers USA) and incubated overnight at 4C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4C. Beads were washed with 500ml buffer for 5 min at 4C with five rounds of washes. After washing, chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 µL 5M NaCl, 2 µL proteinase K were added and samples were incubated on a shaking heat-block for 4 hours at 1000rpm, 65C. This elution procedure was also repeated for chromatin input samples, where 0.5x10^6 cells were used for input as well.
As final step, the DNA was isolated using QIAGEN MinElute PCR purification Kit and eluted in 20 µL elution buffer.
For each ChIP, chromatin of 0.5x10^6 cells was incubated with 254 uL dilution buffer, 12uL protease inhibitor cocktail (25x), and 1µg of H3K4me3 antibody (Cell Signaling Technology, Danvers USA) and incubated overnight at 4C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4C. Beads were washed with 500ml buffer for 5 min at 4C with five rounds of washes. After washing, chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 µL 5M NaCl, 2 µL proteinase K were added and samples were incubated on a shaking heat-block for 4 hours at 1000rpm, 65C. This elution procedure was also repeated for chromatin input samples, where 0.5x10^6 cells were used for input as well.
As final step, the DNA was isolated using QIAGEN MinElute PCR purification Kit and eluted in 20 µL elution buffer.
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