Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 We previously identified T-DMRs using Restriction Landmark Genomic Scanning (RLGS) [19,32], and some of these RLGS T-DMRs were used as internal controls for the quality of IP DNA prior to the array hybridiza- tion. Quantitative PCR analysis of input and IP DNA samples indicated that the IP DNA from the appropriate tissue was enriched for DNA sequences previously shown to have tissue specific DNA methylation (pre- sence or absence of methylated DNA fragments at Pst6 and Pst21 were confirmed prior to the hybridization; data not shown). Additional file 15 shows the results of MeDIP methylation analysis in the regions of four pre- viously identified RLGS T-DMRs (Pst4, Pst10, Pvu4 and Pvu5) that were confirmed by Sequenom MassArray quantitative methylation analysis [32]. These results are consistent with both RLGS analysis and the Sequenom analysis of bisulfite treated DNA [32]. Among the 68 RLGS-TDMRs, 38 overlap with the probes on the array (allowing a distance of 750 bp the window size we used for peak identification), of which 27 overlap with the MeDIP DS-DMRs or T-DMRs. Among the 11 not picked up by MeDIP, 5 are in the repetitive regions and would not be included on the array. Therefore, the sen- sitivity level of our MeDIP in reference to RLGS is 27/ 33 or 82%. Sequenom MassArray quantitative methylation analysis of bisulfite treated DNA was performed as an additional means to confirm DNA methylation peaks obtained by MeDIP/NimbleGen Array for both selected and randomly chosen MeDIP methylated regions. The Sequenom Mas- sArray results were consistent with the MeDIP results in 19 of 23 peaks that were evaluated (18 randomly chosen and 5 selected peaks). An example of the Sequenom Mas- sArray results for a CpGi promoter region is shown in Figure 1, and for a randomly selected non-CpGi promoter region (Additional file 16) indicating the two methods are in agreement. We also interrogated MeDIP data within imprinted regions that are known to be methylated in somatic tis- sues. Almost the entire imprinted region on Chr 7 that includes the Kcnq1 gene is represented in a tiling array (1,390 kb) on the NimbleGen promoter + CpGi array. A 40 kb portion of the Kcnq1 that contains KvDMR is shown in Additional file 2. The KvDMR is an imprinted control region of approximately 2 Kb (boxed region) that is maternally methylated and has previously been shown to be unmethylated in sperm [56]. Consistent with these earlier results, the MeDIP/NimbleGen array results clearly indicate that DNA from the testis is not methylated in this region. Similarly, the imprinting con- trol region associated with Igf2r is also unmethylated in testis [57] (Additional file 17). Since only the maternal allele is methylated in these imprinted regions, MeDIP is capable of detecting methylation in a single allele. Page 14 of 16 Analysis of relationship between T-DMRs/DS-DMRs and gene expression Two gene expression data sets, GSE9954 and GSE13149, that are deemed suitable for our purpose were identified and retrieved from the NCBI GEO database. For GSE9954, we used the normalized expression values pro- vided in GEO by the authors and performed the distribu- tion pattern and statistical analysis of expression for genes associated with all T-DMRs from each tissue. Specifically, for T-DMRs from each of the 3 location categories, i.e., the CpGi promoter, the non-CpGi promoter and intra- genic CpGi, in a tissue, we compared the expression level and distribution pattern of their associated genes in that tissue with that of the same set of genes in three other tis- sues, as well as gene associated T-DMRs in other two loca- tion categories, based on box plot and t-test using the R package. For GSE13149, the raw Cel files were down- loaded from GEO for samples at E15.5, Day0 and adult mice, which presumably correspond to our E15, new born (NB) and adult samples, and were processed for gcRMA and quantile normalization using the BioConductor R page. The expression data in association with liver DS- DMRs was analyzed in a manner similar to T-DMRs. Additional material Additional file 1: E15 and ES T-DMRs. Additional file 2: MeDIP/NimbleGen Promoter + CpGi Array (Tiling region): Methylation analysis of KvDMR region. The scaled log2 ratio of the 40 Kb region on chromosome 7 near KvDMR is shown. The numbers on the top indicate the genomic position. The rectangle indicates the position of the KvDMR that includes two CpGi regions. Two independent tissues were taken from different mice. Previous studies indicate that the KvDMR is methylated in somatic tissue and unmethylated in sperm [57]. Additional file 3: T-DMRs in tiling regions. Additional file 4: Pearson Coefficient and common peak number between replicates. Additional file 5: a: Overlaps between different tissues for DS-DMRs with testis excluded; b:Overlaps between tissues for DMRs shared by all developmental stages in a tissue. Additional file 6: T-DMRs within the Hoxa gene cluster. The MeDIP methylation profile for the Hoxa gene cluster within the 52.08 to 52.20 Mb region on chromosome 6 is shown. The locations of CpGi regions, Hoxa gene transcripts from a1 to a13 and transcription direction are indicated. The numbers on the bottom from 1 to 8 indicate the position of methylation peaks (represented by the log2 ratio) corresponding to those listed in Additional file 7. Additional file 7: Methylation peaks locations in the Hoxa gene cluster. Additional file 8: DNA methylation and alternative promoters for Pcdha genes. Methylation profile for Pcdha genes is shown in a similar manner as in Additional file 2. See also Additional file 9. Additional file 9: Methylation peaks locations in the Protocadherin gene cluster. Additional file 10: Relationship between T-DMRs and expression of their associated genes. The expression of the genes associated with liver unique T-DMRs were compared to that of the same gene set in the three other tissues broken down into the CpGi promoter (CpGi_P), non- CpGi promoter (NCpGi_P) and intra-genic CpGi (Intra_CpGi) groups using