Chromatin Immunoprecipitation Assay Protocol
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Corresponding Organization :
Other organizations : The Alfred Hospital, Baker Heart and Diabetes Institute, Monash University, University of Melbourne
Variable analysis
- Antibodies used for immunoprecipitation: anti-OCT4, anti-H3K4me1, anti-H3K4me3, anti-H3K4me2, anti-H3K27me3, anti-H3K9/K14ac, anti-H4ac, anti-CTCF, anti-SOX2, anti-STAT3, anti-SRF
- Quantification of the immunoprecipitated DNA by qPCR
- Cell crosslinking with 1% formaldehyde for 10 minutes
- Quenching with 0.125M glycine for 10 minutes
- Resuspension of cell pellets in SDS Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0 and proteinase inhibitor cocktail)
- Sonication of chromatin for 30 minutes at 30 second intervals using a Bioruptor
- Pre-clearing of 4 µg of chromatin with Dynabeads protein A (Invitrogen)
- Immunoprecipitation using anti-rabbit IgG (Millipore) as a negative control
- Anti-rabbit IgG (Millipore) as a negative control
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