ChIP-seq was performed by Active Motif (Carlsbad, CA). Briefly, 1×106 MCF7 cells were seeded in 15cm dishes in phenol red-free medium supplemented with 5% CSS for 72 hrs. Cells were pre-treated with vehicle, 10 µM Enza, or 30uM MJC13 for 3 hrs. E2 was then added for 1 hr in continued presence of vehicle, Enza, or MJC13. For AR ChIP-seq, an additional sample was treated with DHT for 4 hrs. The cells were washed with PBS then fixed as per manufacturer’s instructions (Active Motif). AR antibody H-280 (Santa Cruz) or ER antibody HC-20 (Santa Cruz) were utilized. Peak calls were made by MACS2 (23 (link)) with default parameters using the sequence alignments obtained from Active Motif. Motif discovery was performed on 100 base pairs surrounding the peak summit using BioProspector (24 (link)). Patser (25 (link)) was used to determine significant matches to AREs and EREs.
Protocol detail
ChIP-seq Analysis of AR and ER
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Corresponding Organization : University of Colorado Anschutz Medical Campus
Other organizations : Huntsman Cancer Institute, University of Utah
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Variable analysis
independent variables
- Vehicle treatment
- 10 µM Enza treatment
- 30 µM MJC13 treatment
- 4 hrs DHT treatment
- AR binding profile (via ChIP-seq)
- ER binding profile (via ChIP-seq)
- MCF7 cell line
- Phenol red-free medium supplemented with 5% CSS
- Cell seeding density (1×10^6 cells)
- Cell culture duration (72 hrs)
- Antibodies used for ChIP (AR H-280, ER HC-20)
- Peak calling method (MACS2)
- Motif discovery method (BioProspector)
- Motif matching method (Patser)
- None specified
- None specified
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