ChIP-qPCR analysis was performed in triplicate as previously described (22 (link)). Following hormonal treatment as described above, the primary cell cultures were cross-linked for 15 min with 1.5% formaldehyde in PBS and the reaction was quenched using 1.25 M glycine. Cell extracts were collected in PBS and chromatin fragments prepared via sonication. Samples were then pre-cleared over night, followed by immunoprecipitation using either a control IgG or pCREB (ser 133) antibody. After washing, the protein-DNA crosslinks were reversed and the DNA fragments were purified using a QIAquick PCR kit (Qiagen, Valencia, CA). Quantitative RT-PCR was performed using Fast Start Universal Sybr green (Roche Applied Science, Indianapolis, IN) and primers designed to amplify the control region 30kb upstream of Mmp13 TSS, as well as the RL-D2, RL-D4 and RL-D5 enhancer regions of the Tnfsf11 locus.
Protocol detail
ChIP-qPCR Analysis of Transcription Factors
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Antibody
Cell extracts
Chip
Chromatin
Formaldehyde
Glycine
Immunoprecipitation
Mmp13
Primary cell cultures
Primers
Protein dna
Rt pcr
Sybr green
Tnfsf11
Corresponding Organization :
Other organizations : University of Wisconsin–Madison
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Variable analysis
independent variables
- Hormonal treatment
- Enrichment of DNA fragments from the control region 30kb upstream of Mmp13 TSS
- Enrichment of DNA fragments from the RL-D2, RL-D4 and RL-D5 enhancer regions of the Tnfsf11 locus
- Time of cross-linking (15 min)
- Concentration of formaldehyde (1.5%)
- Quenching of the reaction using 1.25 M glycine
- Sonication for chromatin fragment preparation
- Overnight pre-clearing of samples
- Immunoprecipitation using a control IgG or pCREB (ser 133) antibody
- Washing steps after immunoprecipitation
- Reversal of protein-DNA crosslinks
- DNA purification using a QIAquick PCR kit
- Quantitative RT-PCR using Fast Start Universal Sybr green and specific primers
- Positive control: Immunoprecipitation using pCREB (ser 133) antibody
- Negative control: Immunoprecipitation using control IgG
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