e89 Nucleic Acids Research, 2009, Vol. 37, No. 12 genome for several diﬀerent species including human and plants. An indirect approach compares expression analysis of 5-aza-cytidine treated to untreated cells (15). Early methods utilized fragments cloned from CpG island libraries (16). Illumina Inc. has developed an array-like procedure based on their bead platform (17). Several other approaches have adopted more standard array plat- forms utilizing DNA precipitations for human and arabi- dopsis with either methyl-binding proteins or antibodies that recognize methyl cytosine (ChIP-chip) (18–21). Other methods utilize methylation sensitive restriction endonu- cleases with or without fractionation of the genome (22–25). More recently, sequencing methods have been developed using the newest generation sequencers such as the Roche Genome Sequencer FLX (454 Lifesciences technology) (26). Although these methods are likely to replace array-based method they are presently prohibi- tively expensive to analyze the entire genome of large sets of samples. It is more likely that for the time being, many islands will be analyzed for few samples or few islands will be analyzed for many samples. We have developed a method to proﬁle genome-wide methylation that is similar to the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay (27) and MASS, which utilizes the enzyme McrBC (28), but we have made modiﬁcations to increase the methyla- tion detection limits as well as the classes of islands ana- lyzed. We have analyzed cell lines and validated measurements of methylation with bisulﬁte sequencing. We then went on to develop methods, which allow us to analyze tumors with unmatched normals, thereby acces- sing any samples in our tumor collection. This methodol- ogy will be useful to identify methylation events in the ﬁeld of cancer as well as other ﬁelds such as development, aging, imprinting, etc. METHODS Materials Enzymes, MspI, McrBC, T4 DNA ligase, were supplied by new England Biolabs. Primers were supplied by Sigma Genosys. Cot1 DNA and tRNA were supplied by invitro- gen. The Megaprime labeling kit, Cy3-conjugated dCTP, and Cy5-conjugated dCTP were supplied by Amersham- Bioscience. Taq polymerase [Eppendorf mastermix (2.5X)] was supplied by Eppendorf. Centricon YM-30 ﬁlters were supplied by Amicon and formamide was supplied by Amresco. Phenol:chloroform was supplied by Sigma. NimbleGen photoprint arrays were synthesized by NimbleGen Systems Inc. Design of the array was described previously (29). Samples Cell lines, SKBR3, Huh7, PANC1, were acquired through ATCC and grown according to speciﬁed conditions. The cell line chp-skn-1 is a primary ﬁbroblast cell line cultured from a skin sample provided by an anonymous donor, cultured under the following conditions: DMEM + 20% FBS, Penn/Strep, and non-essential amino acids. Tumor DNA from 11 patients with advanced ovarian carcinomas PAGE 2 OF 11 who were treated at the Department of Gynecological Oncology at The Norwegian Radium Hospital (NRH) during the period May 1992 to February 2003 were included in this study. The collection is approved by the Regional ethical review board (Reference No: S-01127). Tumor DNA from 28 patients treated for localized breast cancer at the Norwegian Radium Hospital (NRH) from 1995 to 1998 was included in this project. The samples were collected under an informed consent and the project approved by the local REK/IRB (30). A small number of samples 12 breast tumors and 12 normals and 7 ovarian normals were obtained from The Cooperative Human Tissue Network, a repository of tumor material run by the National Institutes of Health. Methylation array and detection Coverage. Our approach to map genome-wide methyla- tion involves tiling all the predicted CpG islands. All annotated CpG islands were obtained from the UCSC genome browser. These islands were predicted using the published Gardiner-Garden and Frommer (1) deﬁnition and involves the following criteria: length 200 bp, %GC 50%, observed/expected CpG 0.6. There are 26 219 CpG islands in the range of 200–2000 bp in the genome. These islands are well covered by Msp I restriction fragmentation. Arrays were manufac- tured by Nimblegen Systems Inc. using the 390K format to the following speciﬁcations. The CpG island annotation from human genome build 33 (hg17) was used to design a 50-mer tiling array. The 50 mers were shifted on either side of the island sequence coordinates to evenly distribute the island. The 390 K format has 3 67 658 available features which would not ﬁt all islands with a 50-mer tiling. Therefore, we made a cutoﬀ on the islands to be repre- sented based on size, with only CpG islands of size 200–2000 bp being assayed. The array represents classical CpG islands and does not include imprint control regions or other non-island promoters known to be methylated. Background hybridization signal could be high with probes of high GC content since by deﬁnition these probes will be. Therefore, control probes, which are not in an MspI representation, were designed to represent background signal, and these probes were used to calcu- late signal to noise (see Supplementary Figure 2). Array design, probe sequences and further annotation are available on line http://www.ncbi.nlm.nih.gov/geo/, data- set number GSE15801. Sample preparation and hybridization Representations have been described previously (29), with the following changes. The primary restriction endonu- clease used is MspI. After the digestion the following lin- kers were ligated (MspI24mer CAGCATCGAGACTGA ACGCAGCAG, and MspI12mer CGCTGCTGCGTT. The 12 mer is not phosphorylated and does not ligate. After ligation the material is cleaned by phenol chloro- form, precipitated, centrifuged and resuspended. The material is divided in two, half being digested by the endo- nuclease McrBC and the other half being mock digested according to speciﬁcation by New England Biolabs.