C15410003

Manufactured by Diagenode

Sourced in United States

The C15410003 is a laboratory instrument designed for disrupting biological samples. It utilizes high-frequency vibrations to mechanically disrupt cells, tissues, or other biological materials, facilitating efficient extraction of target biomolecules such as DNA, RNA, or proteins. The instrument is suitable for a range of sample types and volumes, making it a versatile tool for various applications in life science research and molecular biology.

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H3K4me3 C15410003 (2 citations)

8 protocols using c15410003

Chromatin Profiling with Histone Modifications

Cited 1 time

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Chromatin immunoprecipitation (ChIP) was performed as previously described (3 (link)) using Protein A or G magnetic beads (Dynabeads, Life Technologies) to capture antibody-chromatin complexes. Antibodies used were anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (C15410003, Diagenode), anti-H3K4ac (07-539, millipore), anti-H3K9ac (ab4441, Abcam), anti-H3K9me3 (05-1242, millipore), H3K27ac (ab4729, Abcam), and anti-H3K27me3 (07-449, millipore).

Handel A.E., Shikama-Dorn N., Zhanybekova S., Maio S., Graedel A.N., Zuklys S., Ponting C.P, & Holländer G.A. (2018). Comprehensively Profiling the Chromatin Architecture of Tissue Restricted Antigen Expression in Thymic Epithelial Cells Over Development. Frontiers in Immunology, 9, 2120.

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Validating Epigenetic Modifications in Synchronized Cells

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We used western blot analysis to validate the Pol II protein level, and H3K4me3 and H3K27me3 modification levels in double thymidine blocked synchronized HaCaT cells (Supplementary Method 4). Primary antibodies for Pol II (C15200004), H3K4me3 (C15410003), and H3K27me3 (C15410195) were obtained from Diagenode.

Hegre S.A., Samdal H., Klima A., Stovner E.B., Nørsett K.G., Liabakk N.B., Olsen L.C., Chawla K., Aas P.A, & Sætrom P. (2021). Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation. Scientific Reports, 11, 18952.

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ChIP-seq for Epigenomic Profiling

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ChIP was performed according to standard protocol [50 (link)] with minor modifications. Paraformaldehyde (1 %) cross-linking was carried out for 10 min followed by the chromatin preparation as described earlier [7 (link)]. Nuclei were re-suspended in ChIP-incubation buffer at a concentration of 20 × 10⁶ cells/mL and sheared (seven cycles with each cycle containing 10 s power on and 10 s interval) using Bioruptor®Plus (B01020001, Diagenode, Liege, Belgium). Sonicated chromatin equivalent of 4 × 10⁶ cells was incubated with relevant antibody overnight at 4 °C. Antibodies against P300 (sc-585x, Santa Cruz Biotechnology, Inc., Dallas, Texas, United States), POLII (MMS-126R-500, Covance, Inc., Princeton, New Jersey, United States), H3K27ac (C15410196, Diagenode), H3K4me1 (C15410194, Diagenode), and H3K4me3 (C15410003, Diagenode) were used. ChIP-seq sample preparation and sequencing was performed according to manufacturer’s instructions (Illumina, San Diego, California, United States) and essentially as described [6 (link), 9 (link), 51 (link)] (http://www.blueprint-epigenome.eu).

Kuznetsova T., Wang S.Y., Rao N.A., Mandoli A., Martens J.H., Rother N., Aartse A., Groh L., Janssen-Megens E.M., Li G., Ruan Y., Logie C, & Stunnenberg H.G. (2015). Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization. Genome Biology, 16, 264.

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Antibody Usage in Protein Analysis

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The following antibodies were used for immunoblotting and immunoprecipitation: polyclonal anti-ZC3H4 antibody, developed against a purified GST-tagged human ZC3H4 fragment (amino acid residues 1114–1315) in this study; polyclonal anti-WDR82 antibody (kindly provided by the Roeder laboratory); anti-CK2α (10992-1-AP, Proteintech); anti-CK2β (ab76025, Abcam); anti-SPT5 (A300-869A for immunoblotting; A300-868A for immunoprecipitation, Bethyl Laboratories); anti-RNAPII (ab26721, Abcam); anti-S5p RNAPII (ab5131, Abcam); anti-S2p RNAPII (ab5095, Abcam); anti-β-actin (TA811000, Origene); anti-phospho-CK2 substrate [(pS/pT)DXE] (8738, Cell Signaling Technology) and anti-FLAG (A8592, Sigma-Aldrich). The following antibodies were used for ChIP-seq analyses: anti-ZC3H4 (this study) and anti-WDR82 (48 (link)), generated in house; anti-CK2α (ab70774, Abcam); anti-SPT5 (sc-133217, Santa Cruz); anti-RNAPII (A300-653A, Bethyl Laboratories); anti-S5p RNAPII (ab5131, Abcam); anti-Sp2 RNAPII (A300-654A, Bethyl Laboratories); anti-H3K4me3 (C15410003, Diagenode); anti-H3K27me3 (9733, Cell Signaling Technology) and anti-H3K36me3 (61101, Active Motif).

Park K., Zhong J., Jang J.S., Kim J., Kim H.J., Lee J.H, & Kim J. (2022). ZWC complex-mediated SPT5 phosphorylation suppresses divergent antisense RNA transcription at active gene promoters. Nucleic Acids Research, 50(7), 3835-3851.

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ChIP-qPCR analysis of histone marks

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Primed cells were harvested on day 6 after crosslinking for Chromatin Immunoprecipitation (ChIP) assays using the MAGnify Chromatin Immunoprecipitation System (Invitrogen) according to manufacturer’s instructions. Chromatin of 200,000 cells was immunoprecipitated with 1 μg of antibody raised against H3k4me3 (Diagenode #C15410003) or H3k27ac (Diagenode #C15410196). Rabbit polyclonal IgG (Diagenode, #C15410206) was used as a negative control. Immunoprecipitated DNA was amplified by quantitative RT-PCR using SYBR green. Enrichment of different histone marks was identified using specific primers for IL-6 and TNFα promoters. The IgG control did not yield a signal in the PCR analysis.

Sohrabi Y., Sonntag G.V., Braun L.C., Lagache S.M., Liebmann M., Klotz L., Godfrey R., Kahles F., Waltenberger J, & Findeisen H.M. (2020). LXR Activation Induces a Proinflammatory Trained Innate Immunity-Phenotype in Human Monocytes. Frontiers in Immunology, 11, 353.

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H3K4me3 ChIP-seq Protocol using RELACS

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The H3K4me3 ChIP-seq was performed using RELACS as previously
described^{70 (link)}.
Briefly, 50,000 β_HI and β_LO cells were
thawed in RELACS lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2%
Igepal, 1× Protease inhibitor cocktail) and the nuclei were isolated
by sonication using the NEXSON procedure^{69 (link)}. To digest the chromatin, 25 μL of
10× CutSmart buffer (NEB), 2.5 μL 100× Protease
inhibitor cocktail and 1 μL of CviKI-1 (5 U/100,000 nuclei, NEB
R0710S) were added. The digestion reaction was incubated overnight at
20°C. End repair and A-tailing was performed, and customized adapters
were ligated to the fragments. Once barcoded, the samples were pooled
together. Chromatin was then sheared by sonication (Covaris E220,
MicroTubes, 5 min, peak power 105, duty factor 2, cycles burst 200). This
chromatin was used for automated ChIP (Diagenode, C15410003) with the
IP-Star Diagenode system. IPs and Inputs were de-crosslinked, DNA was
purified, and libraries were prepared using the NEB Ultra II DNA Library
Prep Kit for Illumina (E7645S and E6440) following the manufacturer’s
instructions. Integrity and size distribution of the samples was assessed
before and after library preparation by running on Fragment Analyzer
(Advanced Analytical).

Dror E., Fagnocchi L., Wegert V., Apostle S., Grimaldi B., Gruber T., Panzeri I., Heyne S., Höffler K.D., Kreiner V., Ching R., Lu T.T., Semwal A., Johnson B., Senapati P., Lempradl A., Schones D., Imhof A., Shen H, & Pospisilik J.A. (2023). Epigenetic dosage identifies two major and functionally distinct β-cell subtypes. Cell metabolism, 35(5), 821-836.e7.

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Extracting and Analyzing Histone Modifications

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100 mg of 21 day-old seedlings were harvested 4 hours after the light onset and flash-frozen in liquid nitrogen. The histones were extracted following the protocol described in Bowler et al. (2004) (link) with the following modifications: the samples were resuspended in 1 mL of buffer 1. After filtration through Miracloth, the samples were centrifuged 20 min at 4000 rpm at 4°C. The pellets were resuspended in 300 μL of buffer 2, centrifuged 10 min at 13000 rpm at 4°C and resuspended in 300 μL of buffer 3 and layered on 300 μL of clean buffer 3. After a 1 h centrifugation at 13000 rpm at 4°C, the pellets were resuspended in 100 μL of nuclei lysis buffer. The protein concentration was assessed using the Qubit protein assay (ThermoFisher Scientific) and all samples were adjusted to the same concentration using nuclear lysis buffer. The immunoblot analysis was performed as described in Hisanaga et al. (2023) (link) using the following antibodies: α-H3K27me3 (C15410195 Diagenode), α-H3K4me3 (C15410003, Diagenode) and α-H3pan (C15200011 Diagenode). The imaging was performed using the Image Studio Lite software (Li-Cor, version 5.2). The intensity of the H3K27me3 and H3K4me3 signals were normalized to the intensity of the H3 signal.

Faivre L., Kinscher N.F., Kuhlmann A.B., Xu X., Kaufmann K, & Schubert D. (2024). Cold stress induces rapid gene-specific changes in the levels of H3K4me3 and H3K27me3 in Arabidopsis thaliana. Frontiers in Plant Science, 15, 1390144.

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ChIP-seq Protocol for Embryonic Epidermal Tissue

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Chromatin immunoprecipitation (ChIP) experiments were performed as previously described [64 (link)] with minor modifications. For each experiment, 50 posterior abdominal epidermes (A5, A6 and A7) of females between 0 and 2h after hatching and 3μg of antibody were used. Results present the mean of three independent experiments for each antibody. Tissue disruption was performed before cell lysis using the FastPrep technology (MP Biomedicals, Lysis matrix D, 20 seconds at 4m/s). Chromatin sonication was performed in a Bioruptor sonifier (Diagenode) (16 cycles of 30'' ON, 30'' OFF, High power). Input and immunoprecipitated DNA were purified with the Ipure kit (Diagenode) in 70μl of water and 4μl were used per qPCR reaction. qPCR experiments were carried out in a CFX96 system (Biorad) using SsoFast EvaGreen Supermix (Biorad). Primers used are listed in S1 Table. Data were normalized against input chromatin or panH3 ChIP. Antibodies used were anti-H3K4me3 (C15410003, Diagenode), anti-H3K27ac (C15410174, Diagenode), anti-panH3 (C15310135, Diagenode). Rabbit IgGs (Diagenode) were used as negative control (Mock).

Gibert J.M., Mouchel-Vielh E., De Castro S, & Peronnet F. (2016). Phenotypic Plasticity through Transcriptional Regulation of the Evolutionary Hotspot Gene tan in Drosophila melanogaster. PLoS Genetics, 12(8), e1006218.

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