ChIP assays were performed as previously described (46 (link)). Briefly, aliquots of 2.5×106 formaldehyde-fixed cells were resuspended in 50 μl nuclei isolation buffer (Abcam, Cambridge, United Kingdom), and chromatin was digested with 15 U MNase (ThermoFisher) for 5 min. at 37ºC. EDTA was added to stop the reaction. After enzymatic digestion, antibodies against Runx1 (ab23980, Abcam), Runx2 (AF2006, R&D Systems, Minneapolis, MN), Runx3 (353604, BioLegend), TET2 (PA5–35847, ThermoFisher), and TET3 (PA5–34431, ThermoFisher) were added to precipitate the sheared chromatin. An IgG isotype antibody (MAB004, R&D Systems) was also added for a background control. After washing steps, protein and DNA complexes were eluted and cross-links were reversed. Purified DNA samples were analyzed by quantitative real time PCR with primers listed in Supp. Table 1 .
Nuclei isolation buffer
Manufactured by Abcam
Nuclei isolation buffer is a solution used to isolate and purify cell nuclei from various biological samples. It facilitates the separation of nuclei from the cytoplasmic components of cells, allowing for the study and analysis of nuclear-specific processes and structures.
Automatically generated - may contain errors
Lab products found in correlation
Mnase, by Thermo Fisher Scientific (2 mentions)
Ab23980, by Abcam (1 mentions)
Mab004, by R&D Systems (1 mentions)
Af2006, by R&D Systems (1 mentions)
Ab5131, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Nbp1 83622, by Novus Biologicals (1 mentions)
2 protocols using nuclei isolation buffer
1
Chromatin Immunoprecipitation (ChIP) Assay
2
Chromatin Immunoprecipitation Assay Protocol
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Aliquots of 2.5 × 106 formaldehyde-fixed cells were resuspended in 50 µl nuclei isolation buffer (Abcam). Chromatin was digested by adding 15 U MNase (ThermoFisher) and incubating at 37°C for 5 min. EDTA was added to stop the reaction. Digested chromatin was diluted in immunoprecipitation buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, and 5 mM sodium butyrate) with EDTA-free protease inhibitors (Millipore) and precleared with Protein G Agarose (Millipore) for 1 h at 4°C. Precleared chromatin was immunoprecipitated overnight at 4°C with antibodies against either ARID5B (NBP1-83622; Novus Biologicals), RNA polymerase II CTD repeat YSPTSPS (phospho-S5; ab5131; Abcam), or histone H3 (dimethyl K9; ab1220; Abcam). Samples were washed and eluted, and cross-links were reversed with a 4-h incubation at 65°C. DNA was precipitated and analyzed by qRT-PCR.
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