MCF-7 breast cancer cells were grown to ~80 to 90% confluence, cross-linked with 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 (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 PADI4. 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.
MCF-7 breast cancer cells were grown to ~80 to 90% confluence, cross-linked with 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 (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 PADI4. 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
Hybridization protocol
see NimbleGen website
Scan protocol
see NimbleGen website
Description
no additional information
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.
