cell line: MCF-7 cell type: breast cancer cells treatment: E2 chip antibody: custom-made rabbit polyclonal ERa antibody
Extracted molecule
genomic DNA
Extraction protocol
MCF-7 breast cancer cells were grown to ~80 to 90% confluence, cross-linked with 10mM DMS in PBS for 10min. at room temp., and 1% paraformaldehyde in PBS for 10 min. at 37°C, and quenched in 125 mM glycine in PBS for 5 min at 4°C. The cells were collected by centrifugation and sonicated in lysis buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation, diluted 10-fold in dilution buffer (0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail), and pre-cleared with protein A-agarose beads. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions with antibodies against ER-alpha. The immunoprecipitated genomic DNA was cleared of protein and residual RNA by digestion with proteinase K and RNase H, respectively. The DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol.
cell line: MCF-7 cell type: breast cancer cells treatment: E2 chip antibody: none
Extracted molecule
genomic DNA
Extraction protocol
MCF-7 breast cancer cells were grown to ~80 to 90% confluence, cross-linked with 10mM DMS in PBS for 10min. at room temp., and 1% paraformaldehyde in PBS for 10 min. at 37°C, and quenched in 125 mM glycine in PBS for 5 min at 4°C. The cells were collected by centrifugation and sonicated in lysis buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation, diluted 10-fold in dilution buffer (0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail), and pre-cleared with protein A-agarose beads. An aliquot of the pre-cleared, chromatin-containing supernatant taken as an input sample. The input DNA was cleared of protein and residual RNA by digestion with proteinase K and RNase H, respectively. The DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol.
Label
Cy3
Label protocol
See the NimbleGen website.
Hybridization protocol
See the NimbleGen website.
Scan protocol
See the NimbleGen website.
Description
Custom-made rabbit polyclonal ERa antibody: Kraus WL, Kadonaga JT, Genes Dev. 1998 Feb 1;12(3): 331-42 (PMID 9450928).
Data processing
Genomic data analysis was performed using the statistical programming language R (R Development Core Team). All data processing scripts are available upon request. The log2 ratio data from each of the arrays was subjected to lowess normalization.
