A detailed DNA copy number profile of this cell line has been previously reported .In order to obtain sufficient material for ChIP-chip, large scale cultures were grown using hyperflask cell culture vessels . Small scale cultures were maintained in T-75 culture flasks for qPCR, microarray expression analysis, co-immunoprecipitation and Western blot experiments. The protocol used was as previously described by Weber et al. with adaptations . Briefly, 5 mg of isolated DNA was sonicated to 400�C800 bp in length. The sonicated DNA was incubated overnight with 10 mg of anti-59 methyl-cytidine antibody to immunoprecipitate purchase MK-2206 methylated sequences. SYBR green qPCR Tubacin biological activity analysis was performed prior to microarray hybridization in order to confirm enrichment of methylated sequences . The methylated H19 locus was used as the control and fold enrichment of this locus was determined relative to the unmethylated H3B locus . Input control and MeDIP DNA samples were labeled using Cy3 and Cy5 random primers, respectively, and co-hybridized to a custom miRNA array and to the HG18 two-Array promoter set from Roche NimbleGen. Arrays were scanned using the GenePix 4000B scanner and analyzed using the Nimblescan software Version 2.4. Normalized log2 ratio data was calculated and a one-sided Kolmogorov- Smirnov test using a sliding window of 750 bp was used to determine whether the probes were drawn from a significantly more positive distribution of intensity log-ratios than those on the rest of the microarray. Each probe was assigned a �Clog10 p-value from the windowed KS test and hypermethylated sites of enrichment were detected by searching for at least two probes with a minimum �Clog10 p-value of two. Peaks within 500 bp of each other were merged and SignalMap version 1.9 was used to visual the resulting data files. DNA sequence data for the processed ChIP regions for each cell line were retrieved from the UCSC database. Phylogenetically conserved sequence between Human and Mouse was selected for motif analysis. The occurrence of DNA binding motifs was assessed in relation to their background frequencies within the sequence tiled on the promoter array. Significance for over- or under-representation was assessed using P-values based on Chi-square test. Transcription factor motif enrichment was assessed by examining occurrence of 482 transcription factor binding motifs , represented by their position-weight-matrices , in the conserved DNA sequences from the various peak datasets.