ChIP Assay Using Chromatrap Spin Columns
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Corresponding Organization :
Other organizations : Brown University, Institute for Systems Biology, Université de Montréal, University of Virginia, University of Rochester, Leiden University Medical Center
Variable analysis
- Amount of extract containing 2 μg of DNA
- Amount of antibody (4 μg)
- Immunoprecipitated DNA
- Cell number (2×10^6 cells)
- Crosslinking with 1% formaldehyde (10 min., room temperature)
- Quenching with glycine
- Washing with ice-cold PBS (containing protease inhibitors)
- Resuspension in hypotonic buffer and incubation for 10 min. on ice
- Nuclei resuspension in lysis buffer
- Sonication using Bioruptor UCD-200 instrument (Diagenode) set to pulse on high (30 sec. followed by 30 sec. rest) for a total time of 10 min.
- Centrifugation to remove debris
- Overnight incubation of immunocomplexes at 4°C with mild agitation
- Washing and elution according to the manufacturer's protocol
- Rabbit IgG
- 1% input
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