Amp pure beads

Manufactured by Beckman Coulter

Sourced in United States

AMP Pure beads are magnetic particles designed for various nucleic acid extraction and purification applications. They provide a convenient and efficient method for isolating and concentrating DNA, RNA, or other biomolecules from complex biological samples.

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Lab products found in correlation

Protein g dynabead, by Thermo Fisher Scientific (13 mentions) Proteinase k, by Thermo Fisher Scientific (9 mentions) Sonifier 250, by Emerson (6 mentions) Nextseq 500 genome analyzer, by Illumina (4 mentions) Rnase a, by Roche (4 mentions) Ab4441, by Abcam (1 mentions) Ab8895, by Abcam (1 mentions) Ab15828, by Abcam (1 mentions) Ab9050, by Abcam (1 mentions) H2ak119ub, by Cell Signaling Technology (1 mentions) Ring1b, by Cell Signaling Technology (1 mentions) H3k4me3, by Merck Group (1 mentions) H3k27ac, by Active Motif (1 mentions) H3k27me3, by Merck Group (1 mentions) E220 system, by Covaris (1 mentions) E220 focused ultrasonicator, by Covaris (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions) Truseq protocol, by Illumina (1 mentions) Bcl2fastq, by Illumina (1 mentions) Casava, by Illumina (1 mentions)

15 protocols using amp pure beads

ChIP-Seq Protocol for Cell Lines and Tissues

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ChIP assays were carried out on D341, D283 and MSCs cultures of approximately 2–5 million cells per sample and per epitope, following the procedures described previously (46 (link), 47 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with antibodies overnight at 4C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After crosslink reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 2-5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer. For primary tissue preparations, 10-30 mg of tumor was first cut on dry ice and chopped on ice with a razor blade. Samples were then resuspended in 1 mL cold PBS and fixed 15 minutes at room temperature by adding formaldehyde to a final concentration of 1%. Glycine was added for 5 minutes at room temperature. Samples were first washed in 1 mL cold PBS and resuspended in 1 mL cold PBS before manual homogenization with a syringe and processing as described above.

Boulay G., Awad M.E., Riggi N., Archer T.C., Iyer S., Boonseng W.E., Rossetti N.E., Naigles B., Rengarajan S., Volorio A., Kim J.C., Mesirov J.P., Tamayo P., Pomeroy S.L., Aryee M.J, & Rivera M.N. (2017). OTX2 activity at distal regulatory elements shapes the chromatin landscape of Group 3 medulloblastoma. Cancer discovery, 7(3), 288-301.

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Chromatin Immunoprecipitation Sequencing Protocol

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ChIP assays were carried out on ∼2–5 million cells per sample and per epitope, following the procedures described previously (Mikkelsen et al, 2007 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with the indicated antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After cross-link reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 1–5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer.
Antibodies used for these studies were SMARCA2/4 (39805; Active Motif), H3K4me1 (ab8895; Abcam), H3K4me3 (07-473; Millipore), H3K9ac (ab4441; Abcam), H3K27ac (39133; Active Motif), H3K27me3 (07-449; Millipore), H3K36me3 (ab9050; Abcam), V5 (ab15828; Abcam), RING1B (5694; Cell Signaling), H2AK119ub (8240; Cell Signaling), RYBP (59451204; Sigma-Aldrich), and USP7 (A300-033A; Bethyl Laboratories).

Boulay G., Cironi L., Garcia S.P., Rengarajan S., Xing Y.H., Lee L., Awad M.E., Naigles B., Iyer S., Broye L.C., Keskin T., Cauderay A., Fusco C., Letovanec I., Chebib I., Nielsen P.G., Tercier S., Cherix S., Nguyen-Ngoc T., Cote G., Choy E., Provero P., Suvà M.L., Rivera M.N., Stamenkovic I, & Riggi N. (2020). The chromatin landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms and dependencies. Life Science Alliance, 4(2), e202000808.

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Chromatin Immunoprecipitation Protocol

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For chromatin immunoprecipitation (ChIP) experiments, prepared cells were collected following 48 h of lentiviral infection and 5-d selection (unless otherwise indicate) with puromycin or blasticidin. Capture of chromatin-bound proteins was performed using standard protocols (Millipore). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at 37 °C, the reaction was quenched by addition of 125 mM glycine for 5 min and then five (for synovial sarcoma cell lines) or ten (for fibroblast cell lines) million fixed cells were used per experiment. Chromatin was fragmented by sonication with a Covaris E220 system and the solubilized chromatin was incubated with a primary antibody overnight at 4 °C to form antibody–chromatin complexes. These complexes were incubated with protein G Dynabeads (Thermo Scientific) for 3 h at 4 °C then the beads were washed three times and eluted. The samples then underwent crosslink reversal, treatment with RNase A (Roche) and treatment with proteinase K (Thermo Scientific) followed by DNA capture with AMP Pure beads (Beckman Coulter).

McBride M.J., Mashtalir N., Winter E.B., Dao H.T., Filipovski M., D’Avino A.R., Seo H.S., Umbreit N.T., St. Pierre R., Valencia A.M., Qian K., Zullow H.J., Jaffe J.D., Dhe-Paganon S., Muir T.W, & Kadoch C. (2020). The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma. Nature Structural & Molecular Biology, 27(9), 836-845.

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Chromatin Immunoprecipitation (ChIP) Protocol

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For chromatin immunoprecipitation (ChIP) experiments, indicated cells were harvested following 48 hr exposure to specified lentivirus and 5 day selection with 2 μg/ml of puromycin. ChIP experiments were performed per standard protocols (Millipore, Billerica, MA) with minor modifications. Briefly, cells were cross-linked for 10 min with 1% formaldehyde at 37°C. This reaction was subsequently quenched with 125 mM glycine for 5 min and 5–10 million fixed cells were used per ChIP experiment. Chromatin from fixed cells was fragmented by sonication with a Covaris E220 and the solubilized chromatin was incubated with the indicated antibody (listed in Table S4) overnight at 4°C. Antibody-chromatin complexes were pulled down by incubation with Protein G-Dynabeads (Thermo Scientific) for 3 hours at 4°C, washed and eluted. The samples then underwent crosslink reversal, RNase A (Roche) treatment, and proteinase K (Thermo Scientific) treatment before the captured DNA was extracted with AMP Pure beads (Beckman Coulter).

McBride M.J., Pulice J.L., Beird H.C., Ingram D.R., D’Avino A.R., Shern J.F., Charville G.W., Hornick J.L., Nakayama R.T., Garcia-Rivera E.M., Araujo D.M., Wang W.L., Tsai J.W., Yeagley M., Wagner A.J., Futreal P.A., Khan J., Lazar A.J, & Kadoch C. (2018). The SS18-SSX Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial Sarcoma. Cancer cell, 33(6), 1128-1141.e7.

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ChIP-seq protocol for transcription factors

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ChIP assays were carried out on VCaP or LNCaP cultures of approximately 2–5 million cells per sample and per epitope. ChIP experiments were performed following the procedures described previously (Boulay et al., 2017 (link)). Briefly, cells were cross-linked for 10 min in 1% formaldehyde at 37 °C. This reaction was subsequently quenched in 125 mM glycine for 5 min. Chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Covaris E220 focused-ultrasonicator (Covaris, Inc). Solubilized chromatin was immunoprecipitated with the indicated antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After crosslink reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter).

Sandoval G.J., Pulice J.L., Pakula H., Schenone M.A., Takeda D.Y., Pop M., Boulay G., Williamson K.E., McBride M.J., Pan J., Pierre R.S., Hartman E., Garraway L.A., Carr S.A., Rivera M.N., Li Z., Ronco L., Hahn W.C, & Kadoch C. (2018). Binding of TMPRSS2-ERG to BAF chromatin remodeling complexes mediates prostate oncogenesis. Molecular cell, 71(4), 554-566.e7.

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Chromatin Immunoprecipitation (ChIP) Assays

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ChIP assays were carried out on A673, SKNMC and MSCs cultures of approximately 2–5 million cells per sample and per epitope, following the procedures described previously (Mikkelsen et al., 2007 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with the indicated antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After crosslink reversal, RNaseA, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 1–5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer.

Boulay G., Sandoval G.J., Riggi N., Iyer S., Buisson R., Naigles B., Awad M.E., Rengarajan S., Volorio A., McBride M.J., Broye L.C., Zou L., Stamenkovic I., Kadoch C, & Rivera M.N. (2017). Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain. Cell, 171(1), 163-178.e19.

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ChIP-qPCR Analysis of Protein-DNA Interactions

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Cell fixation, ChIP, and qPCR were performed as described (62 (link)). ChIP assays were carried out using about 10 million HEK293T cells per sample. Forty-eight hours after transfection, cells were cross-linked for 10 min in 1% formaldehyde at 37°C. This reaction was subsequently quenched in 2.5 M glycine for 5 min. Chromatin from formaldehyde-fixed cells was fragmented to a size range of 200 to 700 bases using a sonicator. Solubilized chromatin was immunoprecipitated with anti-MYC and control (IgG) antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with Protein G Dynabeads (Life Technologies), washed, and then eluted. After cross-link reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). The DNA region of interest was detected using RT-qPCR and the listed primers (table S4).

Uribe M.L., Dahlhoff M., Batra R.N., Nataraj N.B., Haga Y., Drago-Garcia D., Marrocco I., Sekar A., Ghosh S., Vaknin I., Lebon S., Kramarski L., Tsutsumi Y., Choi I., Rueda O.M., Caldas C, & Yarden Y. (2021). TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis. Science signaling, 14(688), eabe6156.

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ChIP Assay Protocol for Protein-DNA Interactions

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For ChIP experiments, cells were harvested under the indicated conditions. ChIP experiments were performed according to standard protocols (Millipore), with minor modifications. Briefly, cells were cross-linked for 10 minutes with 1% formaldehyde at 37°C. This reaction was subsequently quenched with 125 mmol/L glycine for 5 minutes, and 5–10 million fixed cells were used for the ChIP experiments. Chromatin from fixed cells was fragmented by sonication with a Covaris E220, and the solubilized chromatin was incubated with the indicated antibody (listed in Supplementary Table S2) overnight at 4°C. Antibody–chromatin complexes were pulled down by incubation with Protein G-Dynabeads (Thermo Fisher Scientific) for 3 hours at 4°C, washed, and eluted. The samples then underwent crosslink reversal, RNase A (Roche) treatment, and proteinase K (Thermo Scientific) treatment before the captured DNA was extracted using AMP Pure beads (Beckman Coulter).

Neri P., Barwick B.G., Jung D., Patton J.C., Maity R., Tagoug I., Stein C.K., Tilmont R., Leblay N., Ahn S., Lee H., Welsh S.J., Riggs D.L., Stong N., Flynt E., Thakurta A., Keats J.J., Lonial S., Bergsagel P.L., Boise L.H, & Bahlis N.J. (2023). ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma. Blood Cancer Discovery, 5(1), 56-73.

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ChIP Assay for Chromatin-Bound Proteins

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For chromatin immunoprecipitation (ChIP) experiments, prepared cells were harvested following 48 hours of lentiviral infection and 5 day selection (unless otherwise indicate) with puromycin or blasticidin. Capture of chromatin bound proteins was performed using standard protocols (Millipore, Billerica, MA). Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes at 37°C, reaction was quenched by addition of 125 mM glycine for 5 min and then 5 (for synovial sarcoma cell lines) or 10 (for fibroblast cell lines) million fixed cells were used per experiment. Chromatin was fragmented by sonication with a Covaris E220 and the solubilized chromatin was incubated with a primary antibody overnight at 4°C to form antibody-chromatin complexes. These complexes incubated with Protein G-Dynabeads (Thermo Scientific) for 3 hours at 4°C, beads washed 3X and eluted. The samples then underwent crosslink reversal, treatment with RNase A (Roche), and treatment with proteinase K (Thermo Scientific) followed by DNA capture with AMP Pure beads (Beckman Coulter).

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ChIP-seq Assay for Transcription Factors

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ChIP assays were carried out on 5 million cells per sample, following the procedures described previously^{52 (link)}. In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 Sonifier. Solubilized chromatin was immunoprecipitated with 5 μg antibodies against AP-2α (Santa Cruz, sc-12726X), NFIB (Sigma-Aldrich, HPA003956), H3K27ac (Active Motif, 39133), V5 (Cell Signaling, 13202) and FLI1 (Abcam, ab15289) at 4 °C overnight. Antibody–chromatin complexes were pulled down with protein G Dynabeads (Life Technologies, 10004D), washed, and then eluted. After cross-link reversal and RNase (Roche, 43813100) and proteinase K (Invitrogen, 25530049) treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter, A63881). ChIP DNA was quantified with Qubit dsDNA HS Assay kit (Invitrogen, Q32854). ChIP DNA samples were used to prepare sequencing libraries with Ultralow V2 DNA-Seq Library Preparation Kit (NuGEN, 0344NB-A01) and DNA samples were sequenced with the Nextseq 500 Illumina genome analyzer.

Xing Y.H., Dong R., Lee L., Rengarajan S., Riggi N., Boulay G, & Rivera M.N. (2023). DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins. Nature Biotechnology, 42(1), 52-64.

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