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Magna chip protocol

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The Magna ChIP protocol is a laboratory equipment product for chromatin immunoprecipitation (ChIP) assays. It provides a standardized and optimized procedure for isolating and purifying DNA-protein complexes from cell or tissue samples. The core function of this product is to facilitate the identification and analysis of DNA-binding proteins and their associated genomic regions.

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Lab products found in correlation

S220 focused ultrasonicator, by Covaris (2 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Magna chip assay kit, by Merck Group (1 mentions) Amersham imager, by Cytiva (1 mentions)

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ChIP protocol (2 citations)

3 protocols using magna chip protocol

1

ChIP-qPCR Assay for Chromatin Immunoprecipitation

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The procedure was based on the Magna ChIP protocol (Millipore, 17-10085) and Chromatrap Premium ChIP qPCR (Chromatrap, 500115). Cells (2 × 106) were cross-linked in 1% formaldehyde for 10 min at room temperature, harvested by scraping, centrifuged, and resuspended in SDS lysis buffer. After 10 min on ice, lysed cells were diluted 2-fold in ChIP dilution buffer and disrupted with the S220 Focused Ultrasonicator (Covaris) to yield genomic DNA fragments of 300 bp. The samples were immunoprecipitated overnight at 4°C with the indicated antibodies. Immunocomplexes were pulled down with 20 μL of magnetic protein A beads as described in the Magna ChIP protocol. The DNA was recovered by 2 hr digestion with proteinase K at 65°C, followed by a 10-min incubation at 95°C. The DNA was cleaned up by phenol/chloroform extraction and ethanol precipitation and finally resuspended in 30 μL of Tris-EDTA (TE).
The ChIP and input were then used for qPCR with the primers described in Table S2.
Ito T., Teo Y.V., Evans S.A., Neretti N, & Sedivy J.M. (2018). Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage-and Histone Methylation-Dependent Pathways. Cell reports, 22(13), 3480-3492.
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2

ChIP-qPCR Assay for Chromatin Immunoprecipitation

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The procedure was based on the Magna ChIP protocol (Millipore, 17-10085) and Chromatrap Premium ChIP qPCR (Chromatrap, 500115). Cells (2 × 106) were cross-linked in 1% formaldehyde for 10 min at room temperature, harvested by scraping, centrifuged, and resuspended in SDS lysis buffer. After 10 min on ice, lysed cells were diluted 2-fold in ChIP dilution buffer and disrupted with the S220 Focused Ultrasonicator (Covaris) to yield genomic DNA fragments of 300 bp. The samples were immunoprecipitated overnight at 4°C with the indicated antibodies. Immunocomplexes were pulled down with 20 μL of magnetic protein A beads as described in the Magna ChIP protocol. The DNA was recovered by 2 hr digestion with proteinase K at 65°C, followed by a 10-min incubation at 95°C. The DNA was cleaned up by phenol/chloroform extraction and ethanol precipitation and finally resuspended in 30 μL of Tris-EDTA (TE).
The ChIP and input were then used for qPCR with the primers described in Table S2.
Ito T., Teo Y.V., Evans S.A., Neretti N, & Sedivy J.M. (2018). Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage-and Histone Methylation-Dependent Pathways. Cell reports, 22(13), 3480-3492.
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3

Chromatin Immunoprecipitation in HAECs and Muscle

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Chromatin immunoprecipitation was performed in HAECs and mouse gastrocnemius muscle specimens by using the Magna ChIP Assay Kit (Millipore, Billerica, USA), according to the manufacturer's instructions. Briefly, HAECs were fixed for 10 minutes with 37% paraformaldehyde. After stopping cross-linking by addition of 0.1 M glycine, cells were sonicated and centrifuged. ChIP was performed by using 10 μg of anti-BRD4 (PA5-41550, Thermo Fischer Scientific) and equivalent amount of mouse IgG as negative control (Cat.02-6102, Invitrogen). Washes and elution of the IP DNA were performed according to the Magna ChIP protocol (17-610, Millipore, Billerica, USA). ChIP quantifications of gene promoters (THBS1) were performed by real-time PCR (primers are shown in Tables S2 andS3). Quantifications were performed using the comparative cycle threshold method and are reported as the n fold difference in antibody-bound chromatin against the input DNA, as previously reported (8, 39) . Biotechnology, Birmingham, AL, USA). Bands were detected by adding HRP substrate (Luminata Forte, Cat. WBLUF0500, Merck Millipore, Billerica, Massachusetts, USA) and visualized using Amersham Imager 600 (General Electric; Healthcare Europe GmbH, Glattbrugg, Switzerland).
Mohammed S.A., Albiero M., Ambrosini S., Gorica E., Karsai G., Caravaggi C.M., Masi S., Camici G.G., Wenzl F.A., Calderone V., Madeddu P., Sciarretta S., Matter C.M., Spinetti G., Lüscher T.F., Ruschitzka F., Costantino S., Fadini G.P, & Paneni F. (2022). The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1. Antioxidants & redox signaling, 36(10-12).
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