Quantitative PCR (qPCR), immunoblotting, and chromatin immunoprecipitation (ChIP) assay were performed as previously described (15 (link)). For the ChIP assay, C2C12 or primary human myotubes were fixed with 1% formaldehyde and sonicated to produce chromatin lysates. The lysates were precleared with Protein-G agarose beads and immunoprecipitated overnight with antibodies against Ace-H3 (Millipore), Ace-H4 (Millipore), H3K4m3 (Millipore), H3K9m2 (Abcam), or control IgG in the presence of BSA and salmon sperm DNA. The next day, Protein-G agarose beads were added to each immunoprecipitation reaction for 1 h, followed by an extensive wash and reverse crosslink. DNA was eluted from the beads and purified using a PCR Purification kit (Invitrogen) and subsequently analyzed by qPCR using primers located on the proximal Baf60c and Deptor promoters. Primers are available upon request.
Protocol detail
Chromatin Profiling of Muscle Cell Epigenetics
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Agarose
Antibodies
Assay
Baf60c
Chromatin
Chromatin immunoprecipitation
Formaldehyde
Human
Immunoprecipitation
Myotubes
Primers
Protein g
Salmon
Sperm
Corresponding Organization :
Other organizations : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Antibodies used for immunoprecipitation (Ace-H3, Ace-H4, H3K4m3, H3K9m2, control IgG)
- Enrichment of DNA fragments on the proximal Baf60c and Deptor promoters, as measured by qPCR
- C2C12 or primary human myotubes used for chromatin immunoprecipitation (ChIP) assay
- Formaldehyde concentration (1%) used for cell fixation
- Presence of BSA and salmon sperm DNA during immunoprecipitation
- Incubation time for immunoprecipitation (overnight)
- Incubation time for Protein-G agarose beads (1 hour)
- None specified
- Control IgG used for immunoprecipitation
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