MCF-7 breast cancer cells were charcoal-stripped for 3 days
Extracted molecule
genomic DNA
Extraction protocol
MCF-7 breast cancer cells were charcoal-stripped for 3 days, stimulated with 100 nM E2 for 45 min at 37 °C, cross-linked with 1% paraformaldehyde for 10 min at 37°C, and quenched in 125 mM glycine for 5 min at 4°C. The cells were collected by centrifugation and sonicated in lysis buffer (1% 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 an antibody against H3Cit26. The immunoprecipitated genomic DNA and a sample of input material were 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. The material was then amplified with LM-PCR.
Label
cy3
Label protocol
See nimblegen website. Input DNA was labelled with cy3 and ChIP DNA was labelled with cy5.
MCF-7 breast cancer cells were charcoal-stripped for 3 days
Extracted molecule
genomic DNA
Extraction protocol
MCF-7 breast cancer cells were charcoal-stripped for 3 days, stimulated with 100 nM E2 for 45 min at 37 °C, cross-linked with 1% paraformaldehyde for 10 min at 37°C, and quenched in 125 mM glycine for 5 min at 4°C. The cells were collected by centrifugation and sonicated in lysis buffer (1% 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 an antibody against H3Cit26. The immunoprecipitated genomic DNA and a sample of input material were 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. The material was then amplified with LM-PCR.
Label
cy5
Label protocol
See nimblegen website. Input DNA was labelled with cy3 and ChIP DNA was labelled with cy5.
Hybridization protocol
see NimbleGen website
Scan protocol
see NimbleGen website
Description
N/A
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.
