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Simplechip enzymatic chromatin ip kit magnetic beads

Manufactured by Cell Signaling Technology
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The SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) is a laboratory tool designed to facilitate chromatin immunoprecipitation (ChIP) experiments. It provides the necessary reagents and protocols to extract, fragment, and immunoprecipitate chromatin samples using magnetic beads.

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Lab products found in correlation

Anti h3, by Cell Signaling Technology (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Primescript rt master mix, by Takara Bio (2 mentions) Sybr premix ex taq, by Takara Bio (2 mentions) Anti pol 2, by Merck Group (1 mentions) Anti sp1 pthr453, by Abcam (1 mentions) Genomeplex single cell whole genome amplification kit, by Merck Group (1 mentions) Anti p53, by Proteintech (1 mentions) H3k27ac, by Cell Signaling Technology (1 mentions) Normal rabbit igg, by Cell Signaling Technology (1 mentions) Nebnext ultra 2 dna library prep kit, by Illumina (1 mentions) Ab76149, by Abcam (1 mentions) Ab172730, by Abcam (1 mentions) Normal mouse igg, by Merck Group (1 mentions) Tead4, by Merck Group (1 mentions) Normal rabbit igg, by Merck Group (1 mentions) Anti dnmt3b, by Cell Signaling Technology (1 mentions) Hiseq 3000 sequencing system, by Illumina (1 mentions) Accel ngs 2s plus dna library kit, by Integrated DNA Technologies (1 mentions) Formaldehyde, by Merck Group (1 mentions)

Close spelling variants of this product

SimpleChIP Enzymatic Chromatin IP Kit (688 citations) SimpleChIP Kit (67 citations) Magnetic beads (20 citations) Enzymatic Chromatin IP Kit (15 citations) SimpleChIP Enzymatic Chromatin IP Kit with magnetic beads (15 citations) Chromatin IP kit (13 citations) SimpleChIP chromatin IP kit (9 citations) SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (7 citations) Enzymatic Chromatin IP (Magnetic Beads), (4 citations) SimpleChIP Plus Enzymatic Chromatin IP Kit with magnetic beads (4 citations)

23 protocols using simplechip enzymatic chromatin ip kit magnetic beads

1

Chromatin Immunoprecipitation Validation

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ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit Magnetic Beads (Cell Signaling Technology). Cross-linked chromatin was incubated with anti-H3 (positive control), anti-IgG (negative control; Cell Signaling Technology), anti-Sp1 (pThr453) (ab59257; Abcam), and anti–Pol II (2019508; Millipore, Billerica, MA) overnight at 4°C with rotation. The precipitated DNA was quantitated by qPCR and normalized by respective 2% input.
Zhu Y.X., Li C.H., Li G., Feng H., Xia T., Wong C.H., Fung F.K., Tong J.H., To K.F., Chen R, & Chen Y. (2020). LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma. Cellular and Molecular Gastroenterology and Hepatology, 10(4), 811-828.
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2

ChIP Assay Using Enzymatic Shearing

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For ChIP, the ‘SimpleChIP Enzymatic Chromatin IP Kit (magnetic Beads)’ (Cell Signaling Technology, via NEB, Frankfurt a. M., Germany) was used according to the protocol. Briefly, 1 × 107 cells were cross-linked by formaldehyde and chromatin was sheared enzymatically. For each ChIP, 30 μg Dynabeads and 5 μg of the SOX2 antibody were used. As control, an antibody against rabbit IgG was included. 2% of sheared chromatin was used as input control. Antibody binding to target complexes was performed overnight at 4°C under constant agitation. Isolation of antibody-target-complexes by Dynabeads was performed at 4°C for 2 h under rotation. Afterwards, DNA was reverse-crosslinked, cleaned up by spin columns (included in the ChIP kit) and amplified with the ‘GenomePlex Single Cell Whole Genome Amplification Kit’ (Sigma Aldrich). Amplified DNA was purified by PCI precipitation and analyzed by qPCR. In qPCR, oligonucleotides were used to amplify a PCR-fragment around the SOX2 binding site (on target) and a PCR-fragment within the same gene, but without a SOX2 binding site (off target). Each qPCR analysis was performed in 3 technical replicates. For antibody and primer details see Tables S1 and S2.
Nettersheim D., Heimsoeth A., Jostes S., Schneider S., Fellermeyer M., Hofmann A, & Schorle H. (2016). SOX2 is essential for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate. Oncotarget, 7(30), 47095-47110.
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3

ChIP-qPCR Analysis of p53 Binding

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Our experiments were conducted using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, MA, United States). Anti-p53 (Proteintech, #10442-1-AP) was used. SimpleChIP® universal qPCR Master Mix (CST, #88989) was used for qPCR. The primers for ChIP-qPCR were as follows: 5′- ACA​TGT​TGA​GCT​CTG​GCA​TAG​A-3′ (forward) and 5′-ggg​gtc​ttt​aga​ggt​ctc​ctg​t-3′ (reverse). Relative enrichment was calculated by normalizing to IgG immunoprecipitation. In the end, agarose gel electrophoresis was used to analyze the PCR products.
Guan L., Yang Y., Lu Y., Chen Y., Luo X., Xin D., Meng X., Shan Z., Jiang G, & Wang F. (2023). Reactivation of mutant p53 in esophageal squamous cell carcinoma by isothiocyanate inhibits tumor growth. Frontiers in Pharmacology, 14, 1141420.
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4

Chromatin Immunoprecipitation (ChIP) Assay

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The SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling, #9003) was used for all chromatin immunoprecipitation (ChIP) assays. In brief, 786O cells were crosslinked using 1% formaldehyde followed by glycine. Cells were washed with ice-cold PBS (×2), scraped and collected by centrifugation. Prepared nuclei were used MNase mediated chromatin digestion after optimization. For optimization, increasing concentrations of MNase were used followed by RNAse A and Proteinase K digestion and DNA isolation. For ChIP assays, 10ug chromatin was used. Diluted chromatin was combined with 10ul of an antibody against Normal Rabbit IgG (1ul, Cell Signaling, #2729), H3K27ac (Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173) or PBRM1 (Cell Signaling, PBRM1/BAF180 (E9X2Z) Rabbit mAb #89123) overnight. Resuspended ChIP-Grade Protein G Magnetic Beads (CS, #9006) were added and incubated for 2hours at 4°C. Beads were washed with a low salt (×3) and high salt (×1) washing buffer. Chromatin was eluted in elution buffer for 30mins at 65°C and cross-linking was reversed via proteinase K (overnight at 65°C). DNA was subsequently collected per manufacturer’s instructions.
For next-generation sequencing, ChIP-seq libraries were prepared from 10 ng of ChIP and input DNAs with the NEBNext Ultra II DNA library Prep Kit for Illumina using the manufacturer’s protocol.
Brooks R., Monzy J., Aaron B., Zhang X., Kossenkov A., Hayden J., Keeney F., Speicher D.W., Zhang L, & Dang C.V. (2022). Circadian lncRNA ADIRF-AS1 binds PBAF and regulates renal clear cell tumorigenesis. Cell reports, 41(3), 111514.
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5

ChIP-seq protocol for c-Jun

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ChIP was conducted by the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads; Cat No. 9003, Cell Signaling Technology) [36 ]. Sequences for primer 1 (with a c-Jun binding site) denoted: 5′-CCAGAGAGGCAGAGAACATA-3’ (forward) and 5′-CATGATGGGAAGCTGGAGTA-3’ (reverse). Meanwhile, an anti-c-Jun (1:100; Cat No. 9165, Cell Signaling Technology) antibody was employed.
Wu L., Du Y., Wang L., Zhang Y, & Ren J. (2024). Inhibition of METTL3 ameliorates doxorubicin-induced cardiotoxicity through suppression of TFRC-mediated ferroptosis. Redox Biology, 72, 103157.
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6

Quantitative Gene Expression and ChIP Analysis

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Total RNA was isolated using TRIzol reagent (Invitrogen, #15596018), and 1 μg of RNA was subjected to cDNA synthesis using PrimeScript RT Master Mix (Takara, #RR036). Quantitative real-time PCR (qPCR) was performed using SYBR Premix Ex Taq (Takara, #RR420) following the manufacturer’s instructions. The expression levels of the indicated mRNAs were calculated using the 2−ΔΔCt method and were normalized to internal control GAPDH. Chromatin immunoprecipitation (ChIP) assays were performed using SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads) (Cell Signaling Technology, #9003 S) according to the manufacturer’s instructions. Quantitative results are displayed as corresponding fold change, and anti-rabbit IgG or anti-mouse IgG were used as a negative control. Primers used for qPCR and ChIP assays are listed in Supplementary Tables S7.
Liu Y.Y., Liu H.Y., Yu T.J., Lu Q., Zhang F.L., Liu G.Y., Shao Z.M, & Li D.Q. (2022). O-GlcNAcylation of MORC2 at threonine 556 by OGT couples TGF-β signaling to breast cancer progression. Cell Death and Differentiation, 29(4), 861-873.
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7

Chromatin Immunoprecipitation of HOXA3

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The ChIP was achieved using SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads; Cell Signaling Technology, Danvers, MA, USA). Briefly, proteins and DNA were cross-linked with 37% formaldehyde for 10 min. AC16 cells were added into lysis buffer for 5 min. Then, cell lysates were dealt with pulses ultrasonication to break nuclear membrane and shear DNA into fragments with 150–900 bp fragments. The final lysates were incubated with 10 μg HOXA3 antibody (cat. no. ab230879; Abcam, Inc., Cambridge, MA, USA), 2 μg nonspecific immunoglobulin G (IgG) antibody, and 10 μg Histone H3 antibody at 4 °C overnight. In all, 10-μl chromatin was used as input control, Histone H3 was used as positive control, and rabbit anti-IgG was used as negative control. The precipitated DNAs were subjected to PCR using a primer pair specific for NLRP3 (Forward: 5′-gagctgaccgtcgtctttga-3′; Reverse: 5′-aaccagctacaaaaagcatggat-3′). The amplified fragments were analyzed by 1.5% (w/v) agarose gel analysis and verified by DNA sequencing.
Li Z., Xu H., Liu X., Hong Y., Lou H., Liu H., Bai X., Wang L., Li X., Monayo S.M., Mokembo J.N., Jha N.K., Yang B, & Zhang Y. (2020). GDF11 inhibits cardiomyocyte pyroptosis and exerts cardioprotection in acute myocardial infarction mice by upregulation of transcription factor HOXA3. Cell Death & Disease, 11(10), 917.
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8

Chromatin Immunoprecipitation of 5-FU Treated Cells

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JSC15-3 cells were treated with 3 µmol/L 5-FU or DMSO as controls. Cells (2 × 107) were fixed with 1% formaldehyde for 10 minutes. Then, 10 × glycine solution was added to the cells, followed by incubation for 5 minutes at room temperature. After washing with cold PBS, the cells were collected using PBS containing a 200 × dilution of a protein inhibitor cocktail. The cells were subjected to chromatin immunoprecipitation (ChIP) using a SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads; Cell Signaling Technology) in accordance with the manufacturer's instructions. Detailed procedures for ChIP-PCR and chromatin immunoprecipitation sequencing (ChIP-seq) data analysis are described in Supplementary Materials and Methods.
Lee J., Mashima T., Kawata N., Yamamoto N., Morino S., Inaba S., Nakamura A., Kumagai K., Wakatsuki T., Takeuchi K., Yamaguchi K, & Seimiya H. (2024). Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer. Cancer Research Communications, 4(5), 1307-1320.
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9

Chromatin Immunoprecipitation (ChIP) Assay

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ChIP assays were performed using a SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, #9003) according to the manufacturer’s instructions. IgG was used as a negative control, and all data are presented as corresponding fold change relative to Input. The primers used for ChIP-qPCR analysis are listed in Supplementary Table S5.
Zhang Y.L., Deng L., Liao L., Yang S.Y., Hu S.Y., Ning Y., Zhang F.L, & Li D.Q. (2022). Chromatin complexes subunit BAP18 promotes triple-negative breast cancer progression through transcriptional activation of oncogene S100A9. Cell Death & Disease, 13(4), 408.
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10

ChIP Assay for Transcription Factor Binding

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ChIP experiments were performed using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads; Cell Signaling Technology, #9003) according to the manufacturer’s instructions. L132 cells were fixed in culture medium containing 1% formaldehyde at room temperature for 10 min to cross-link proteins and DNA. Then, cells were lysed and incubated with 0.5 μl micrococcal nuclease for 20 min at 37°C to digest DNA to 200–500 bp. The mixture was then immunoprecipitated with 2.5 μg NRF1 antibody (ab34682, Abcam) or a negative control IgG at 4°C overnight. The purified DNA was amplified by qRT-PCR with primers designed as follows: RELA (−1394/−1195) forward 5′-ACAGCCTCAGGAAGCCAAAA-3′, reverse 5′-CCTCGGCGGGGATTTTCC-3′; RELA (−1223/−924) forward 5′-CCAGCGTCTGGGGAAAATC-3′, reverse 5′-CCCTCGCGTGGGAGTT-3′; RELA (−823/−525) forward 5′-CTCCTAACGCTGAGGAAGCC-3′, reverse 5′-CGAGGACGTCAGAGTGGAGA-3′; CYCS forward 5′-TTCCTGTCCGACTGTGGTGT-3′, reverse 5′-GGCGGTCTTGTAGTTCTTGATT-3′.
Wang X., Huang L., Jiang S., Cheng K., Wang D., Luo Q., Wu X, & Zhu L. (2021). Testosterone attenuates pulmonary epithelial inflammation in male rats of COPD model through preventing NRF1-derived NF-κB signaling. Journal of Molecular Cell Biology, 13(2), 128-140.
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