RNA was isolated using Promega SV total RNA purification kits. DNA was isolated from frozen tissue samples following homogenisation using a Retsch TissueLyser in the presence of 600 uL of chilled Nucleic Acid Solution. DNA was then isolated following the recommended protocol of the kit. DNA fully methylated http://www.selleckchem.com/products/Imatinib-Mesylate.html at CpG sites, CpGenome DNA, was purchased from Millipore. DNA from pooled peripheral blood of healthy individuals was purchased Inhibitors,Modulators,Libraries from Roche Applied Science. Gene expression arrays Levels of gene expression in CRC cell lines with or with out d Aza and/or TSA treatment were determined using Affymetrix Exon 1. 0ST gene chips. cDNA was prepared and labelled using the High Capacity cDNA Reverse Transcription Kit from Applied Biosystems and gene chip hybridisation and washing done according to Affymetrix protocols detailed in the GeneChip Whole Inhibitors,Modulators,Libraries Transcript Sense Target Label ing Assay Manual P/N 701880 Rev.

4. Microarrays were processed and analysed using R/Bioconductor. Arrays were normalized using robust multiarray normalization, implemented in the simpleaffy package. Probesets with Inhibitors,Modulators,Libraries differential expression within cell lines were identified using limma. Genome wide DNA methylation analysis Genome wide DNA methylation analysis using SuBLiME has been described previously. Libraries of SuBLiME captured Inhibitors,Modulators,Libraries DNA from three cell lines and from wbc DNA were sequenced using ABI SOLiD 3 chemistry and reads aligned to the genome. Cytosines in these fragments were counted and the summed counts across reads used to identify sites that showed statistically significant elevated methylation, as determined by the edgeR R/Bio conductor package.

Bisulfite Tag measures methylation at TaqI and MspI sites across the genome. Briefly, the method relies on cutting of gen omic DNA with TaqI and MspI, enzymes that both cut DNA independently of methylation Inhibitors,Modulators,Libraries at the central CG of their recognition sites. Following restriction enzyme diges tion the hepatocellular carcinoma DNA was treated with sodium bisulfite without de naturing the double stranded fragments. Thus only the two base overhang reacts with bisulfite, with unmethylated cy tosines being converted to uracils and methylated cytosines remaining unconverted. Separate linkers with appropriate matching overhangs were ligated to the bisulfite con verted ends, allowing separate amplification of popula tions representing methylated and unmethylated DNA. Following labelling with either Cy3 or Cy5 dyes, meth ylated and unmethylated fractions were hybridized with Nimblegen 720 K Promoter tiling arrays. Arrays were scanned using the Axon GenePix 4000b and the Perkin Elmer ScanRI and methylation at indi vidual TaqI or MspI sites determined as described in Additional file 1.