cell line: MCF-7 cell type: breast cancer cells treatment: ethanol control chip antibody: goat polyclonal JNK1 antibody antibody vendor: Santa Cruz antibody catalog #: sc-474
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 JNK1. 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: ethanol control 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. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions with antibodies against JNK1. 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.
Label
Cy3
Label protocol
See the NimbleGen website.
Hybridization protocol
See the NimbleGen website.
Scan protocol
See the NimbleGen website.
Description
See the Nimblegen website
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.
