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Chip dna clean and concentrator

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The ChIP DNA Clean and Concentrator is a laboratory equipment designed to purify and concentrate DNA samples from chromatin immunoprecipitation (ChIP) experiments. The core function of this product is to efficiently remove contaminants and salts from ChIP-derived DNA, while recovering the target DNA fragments.

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Lab products found in correlation

Dynabead, by Thermo Fisher Scientific (5 mentions) Lmna antibodies, by Santa Cruz Biotechnology (2 mentions) X chip protocol, by Abcam (2 mentions) Protein g dynabead, by Thermo Fisher Scientific (2 mentions) Rna pol 2, by Santa Cruz Biotechnology (1 mentions) Erα s305 p, by Merck Group (1 mentions) Nbp1 87952, by Novus Biologicals (1 mentions) Bead beater, by Biospec (1 mentions) Cab1001, by Thermo Fisher Scientific (1 mentions) E220 sonicator, by Covaris (1 mentions) Protease inhibitor, by Roche (1 mentions) 0.5 mm glass bead, by Biospec (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)

12 protocols using chip dna clean and concentrator

1

LMNA Chromatin Immunoprecipitation Protocol

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LMNA antibodies (Santa Cruz Biotechnology; sc-6215 and sc-7292) was incubated with Dynabeads (Life Technology; 10003D) for 12 hr at 4 °C. A small portion of the crosslinked, sheared chromatin was saved as the Input, and the remainder was employed for the immunoprecipitation using antibody-conjugated Dynabeads. After overnight incubation at 4 °C, the incubated beads ware rinsed with a sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF), a high salt buffer (50 mM Hepes pH 7.9 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF), and a LiCl buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF). The washed beads were incubated with an elution buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) for 1 hr at 65 °C and then de-crosslinked with 5 M NaCl overnight at 65 °C. The immunoprecipitated DNA was treated with Rnase A and Proteinase K, and purified by ChIP DNA clean and concentrator (Zymo Research; D5205). The raw sequencing data were analyzed as previously described39 (link).
Lee J., Termglinchan V., Diecke S., Itzhaki I., Lam C.K., Garg P., Lau E., Greenhaw M., Seeger T., Wu H., Zhang J.Z., Chen X., Gil I.P., Ameen M., Sallam K., Rhee J.W., Churko J., Chaudhary R., Chour T., Wang P.J., Snyder M.P., Chang H.Y., Karakikes I, & Wu J.C. (2019). Activation of PDGF Pathway Links LMNA Mutation to Dilated Cardiomyopathy. Nature, 572(7769), 335-340.
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2

ChIP-seq protocol for MCF-7 cells

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Chromatin immunoprecipitation assays were performed as described before with some revisions (Stender et al., 2010 (link)). MCF-7 cells were crosslinked with 2 mM disuccinimidyl glutarate for 30 minutes prior to 10 minutes of 1% formaldehyde. The antibodies used in these studies were: ERα (sc-543, Santa Cruz Biotechnologies); p65 (sc-372, Santa Cruz Biotechnology); RNA Pol II (sc-900, Santa Cruz Biotechnology); SRC3 (sc-9119, Santa Cruz Biotechnology); and ERα-S305-P (05–922R, Millipore). For the precipitations protein A Dynabeads (10003D, Invitrogen) were coated with antibody prior to pulldown and excess antibody was washed away. Pulldowns occurred while rotating for 16 hours at 4C. Beads were then washed with with TSE I (20 mM Tris/HCl pH 7.4@20°C, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), twice with TSE III (10 mM Tris/HCl pH 7.4@20°C, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1 mM EDTA), and twice with TE followed by elution from the beads using elution buffer (0.1 M NaHCO3, 1% SDS). Elutions were subsequently de-crosslinked overnight at 65° C and DNA was purified using ChIP DNA Clean and Concentrator (Zymo Research) and DNA was either used for qPCR or prepared into libraries for high-throughput sequencing. The primer sequences used in this study are supplied as a supplemental table.
Stender J.D., Nwachukwu J.C., Kastrati I., Kim Y., Strid T., Yakir M., Srinivasan S., Nowak J., Izard T., Erumbi R., Carlson K.E., Katzenellenbogen J.A., Yao X.Q., Grant B.J., Leong H.S., Lin C.Y., Frasor J., Nettles K.W, & Glass C.K. (2017). Structural and molecular mechanisms of cytokine-mediated endocrine resistance in human breast cancer cells. Molecular cell, 65(6), 1122-1135.e5.
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3

Quantifying CTCF and AHCTF1 Chromatin Binding

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The cells were fixed with 1% freshly prepared formaldehyde solution as described previously38 (link). DNA-protein complexes were immune-purified with antibodies against CTCF (Cell Signaling Technology; CS 2899 S; 20 μl per 10 μg of chromatin DNA), TCF4 (rabbit monoclonal, Cell Signaling Technology, 2569S; 10 μl per 10 μg of chromatin DNA), ß-catenin (Cell Signaling, 8480S; 20 μl per 10 μg of chromatin DNA) or AHCTF1 (Novusbio, NBP1-87952; 2 μg per 6.25 μg of chromatin DNA) using Dynabeads protein G (Thermo Sciences, 10004D)21 (link). Following purification of the ChIP DNA (ChIP DNA Clean and Concentrator; Zymo Research, D5205), the associations between the target loci and CTCF and AHCTF1 were quantified by standard qPCR analysis using primer sequences and PCR conditions, as previously described7 (link) (Supplementary Table I).
Chachoua I., Tzelepis I., Dai H., Lim J.P., Lewandowska-Ronnegren A., Casagrande F.B., Wu S., Vestlund J., Mallet de Lima C.D., Bhartiya D., Scholz B.A., Martino M., Mehmood R, & Göndör A. (2022). Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site. Nature Communications, 13, 204.
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4

Chromatin Immunoprecipitation (ChIP) Protocol

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ChIP was performed using the standard protocol (46 (link)). Briefly, the frozen cell pellets were lysed in cold FA lysis buffer with protease inhibitors (Roche) and 0.5 mm glass beads (BioSpec Products) in the Bead Beater (Biospec). The insoluble chromatin obtained from pelleting the WCE was further sheared using the Covaris E220 sonicator. The sheared chromatin was determined to be in the range of 150–600 bp. Immunoprecipitation of TAP- and Myc-bound DNA was performed by incubating the chromatin extracts with 10 μg of polyclonal anti-TAP antibody CAB1001 (Thermo Scientific) and polyclonal anti-rabbit Myc-antibody (Sigma) respectively, bound to magnetic Protein G Dynabeads (Life Technologies) overnight at 4°C. Washes were performed as described in the protocol and the crosslinks were reversed and treated with Proteinase K overnight to obtain ChIP DNA. The DNA was purified using the ChIP DNA Clean and Concentrator (Zymo Research). ChIP assays using anti-CTD antibodies, reverse crosslinking and purification of DNA were performed as described (44 (link)).
Chereji R.V., Bharatula V., Elfving N., Blomberg J., Larsson M., Morozov A.V., Broach J.R, & Björklund S. (2017). Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly. Nucleic Acids Research, 45(15), 8806-8821.
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5

ChIP-qPCR Protocol for STAT1 Binding

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ChIP assays were performed as described before (Stender et al., 2017 (link)). Cells were crosslinked with 2 mM disuccinimidyl glutarate for 30 min prior to 10 min treatment with 1% formaldehyde. The antibodies used in these studies were: STAT1 (sc-345, Santa Cruz Biotechnology). For the precipitations protein A Dynabeads (10003D, Invitrogen) were coated with antibody prior to pulldown and excess antibody was washed away. Pulldowns occurred while rotating for 16 hr at 4°C. Beads were then washed with TSE I (20 mMTris/HCl pH 7.4 at 20°C, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), twice with TSE III (10 mM Tris/HCl pH 7.4 at 20°C, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1 mM EDTA), and twice with TE followed by elution from the beads using elution buffer (0.1 M NaHCO3, 1% SDS). Elutions were subsequently de-crosslinked overnight at 65°C and DNA was purified using ChIP DNA Clean and Concentrator (Zymo Research) and DNA was used for qPCR. The primer sequences used in this study are:
Cohen Katsenelson K., Stender J.D., Kawashima A.T., Lordén G., Uchiyama S., Nizet V., Glass C.K, & Newton A.C. (2019). PHLPP1 counter-regulates STAT1-mediated inflammatory signaling. eLife, 8, e48609.
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6

Chromatin Immunoprecipitation (ChIP) Protocol

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Preparation of cell lysates for chromatin immunoprecipitation (ChIP) assays was performed according to the X-ChIP protocol (Abcam) with modifications [5 (link)]. 2.5–10 μg of antibodies were used for immunoprecipitation. ChIP-DNA was purified using DNA purification columns (ChIP DNA Clean and Concentrator, Zymo Research, USA) and eluted with 40 μl TE-buffer. DNA was analyzed by quantitative PCR. Antibodies and ChIP-PCR primers are given in S2 File. DNA recovery was calculated as percent of the input, bars represent the standard deviation from at least four independent determinations. ChIP-values of histone modification were corrected for nucleosome density using values gathered by a Histone 3 ChIP.
Kohrs N., Kolodziej S., Kuvardina O.N., Herglotz J., Yillah J., Herkt S., Piechatzek A., Salinas Riester G., Lingner T., Wichmann C., Bonig H., Seifried E., Platzbecker U., Medyouf H., Grez M, & Lausen J. (2016). MiR144/451 Expression Is Repressed by RUNX1 During Megakaryopoiesis and Disturbed by RUNX1/ETO. PLoS Genetics, 12(3), e1005946.
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7

LMNA Chromatin Immunoprecipitation Protocol

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LMNA antibodies (Santa Cruz Biotechnology; sc-6215 and sc-7292) was incubated with Dynabeads (Life Technology; 10003D) for 12 hr at 4 °C. A small portion of the crosslinked, sheared chromatin was saved as the Input, and the remainder was employed for the immunoprecipitation using antibody-conjugated Dynabeads. After overnight incubation at 4 °C, the incubated beads ware rinsed with a sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF), a high salt buffer (50 mM Hepes pH 7.9 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF), and a LiCl buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF). The washed beads were incubated with an elution buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) for 1 hr at 65 °C and then de-crosslinked with 5 M NaCl overnight at 65 °C. The immunoprecipitated DNA was treated with Rnase A and Proteinase K, and purified by ChIP DNA clean and concentrator (Zymo Research; D5205). The raw sequencing data were analyzed as previously described39 (link).
Lee J., Termglinchan V., Diecke S., Itzhaki I., Lam C.K., Garg P., Lau E., Greenhaw M., Seeger T., Wu H., Zhang J.Z., Chen X., Gil I.P., Ameen M., Sallam K., Rhee J.W., Churko J., Chaudhary R., Chour T., Wang P.J., Snyder M.P., Chang H.Y., Karakikes I, & Wu J.C. (2019). Activation of PDGF Pathway Links LMNA Mutation to Dilated Cardiomyopathy. Nature, 572(7769), 335-340.
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8

Stat3 Chromatin Immunoprecipitation Assay

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ChIP assay was performed as previously described with some modifications30 . Briefly, GBM cells were incubated with IL-6 for 24 h with or without Tocilizumab. Following crosslinking and cell lysis, chromatin was sonicated to generate fragments of about 1000 bp. Sonicated chromatin (10%) was kept as the input control material, the rest was incubated with antibodies against Stat3 (79D7, Cell Signaling) followed by incubation with Protein G Dynabeads (Invitrogen). All samples were treated with RNase A and Proteinase K and purified using ChIP DNA Clean and Concentrator (Zymo Research, Irvine, CA) before amplification. DNA was amplified using primers flanking the Stat site (5′-GTACATGATAGAGGAGCCACCA-3′ and 5′-GCAACTTTGCAACTTGGTCTT-3′) and primers of a region 9.5 kb upstream from the transcription start site (5′-GAAGGACCTCTTCAAGGAGA-3′ and 5′-GGATTGTCTTGGCTATACGG-3′) as a negative control. Data were calculated as the percentage of input DNA.
Vidal V., Gutierrez O., Talamillo A., Velasquez C, & Fernandez-Luna J.L. (2021). Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway. Scientific Reports, 11, 16196.
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9

Chromatin Immunoprecipitation and Quantification

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Cells were double-cross-linked with 2 mM EGS for 30 min and then 1% formaldehyde for 5 min. Cells were lysed in RIPA0 (10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate) containing, in addition, 0.25% Sarkosyl, 1 mM DTT, and 1× protease inhibitor (Calbiochem), and chromatin was solubilized and sheared by sonication. NaCl (5 M) was added to solubilized chromatin recovered after centrifugation to a final concentration of 0.3 M. The solubilized chromatin was incubated with anti-PRDM16 antibody (SDIX) immobilized on protein G-conjugated magnetic beads (Dynabeads Protein G, Invitrogen) for 6 h at 4°C. The beads were washed sequentially (twice each) with RIPA0.3 (RIPA0 containing 0.3 M NaCl), RIPA0, LiCl wash buffer (10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate), and TE buffer (pH 8.0). Next, beads were incubated with elution buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, 1% SDS) for 6 h at 65°C. After treatment with RNase A and Proteinase K, the eluates were purified using ChIP DNA Clean and Concentrator (Zymo Research). Precipitated DNA was quantified by real-time PCR as described above. PCR was done in triplicate, and data are shown as percentage of input. Primers used for real-time PCR are described in Supplemental Table 2.
Iida S., Chen W., Nakadai T., Ohkuma Y, & Roeder R.G. (2015). PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1. Genes & Development, 29(3), 308-321.
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10

ChIP DNA Purification and Analysis

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Antibodies were used as described above. Each antibody was incubated with Dynabeads (Life Technology; 10003D) for 12 hr at 4 °C. A small portion of the crosslinked, sheared chromatin was saved as Input and the remainder was employed for the immunoprecipitation using antibody conjugated Dynabeads. After overnight incubation at 4 °C, incubated beads were rinsed with sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF), high salt buffer (50 mM Hepes pH 7.9 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF) and LiCl buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF). Washed beads were incubated with elution buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) for 1 hr at 65 °C and de-crosslinked with 5 M NaCl for overnight at 65 °C. Immunoprecipitated DNA was treated with Rnase A and Proteinase K and purified by ChIP DNA clean and concentrator (Zymo Research; D5205).
Lee J., Shao N.Y., Paik D.T., Wu H., Guo H., Termglinchan V., Churko J., Kim Y., Kitani T., Zhao M.T., Zhang Y., Wilson K.D., Karakikes I., Snyder M.P, & Wu J.C. (2018). SETD7 drives cardiac lineage commitment through stage-specific transcriptional activation. Cell stem cell, 22(3), 428-444.e5.
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