Additional Supporting Information may be found in the online version of this article.

sc-12-0684_SupplFigure1.pdf618KFigure S1: (A) Validation of mass spectrometry-based quantification of 5-methylcytosine (5mC) and 5- hydroxymethylcytosine (5hmC) levels on hypomethylated Np95-deficient ES cells as a biological model, in addition to careful calibration and validation with nucleotide standards. Total 5mC levels are reduced to 30% of wild-type ES cells, and accordingly 5hmC levels drop to very similar levels as expected if 5hmC requires 5mC as a substrate. (B) Expression analysis of the Tet enzymes that confer conversion of 5mC to 5hmC in the four distinct stem cell types, as well as their in vivo tissue counterparts. Low 5hmC levels in extraembryonic stem cells (TS and XEN) correlates with very low Tet1 and Tet2 expression levels, which are the 2 enzymes closely associated with the pluripotent state of ES cells. (C) Methylation analysis by bisulphite sequencing of minor satellite repeats, compared to major satellites as shown in Fig. 1D. Average methylation levels of at least 10 clones each are shown. (D, E) Sequenom analysis of L1 (D) and IAP (E) methylation levels in stem cells and E7.5 in vivo tissues. Data is shown as mean ± SEM.
sc-12-0684_SupplFigure2.pdf537KFigure S2: (A) Valdidation of MeDIP procedure by qPCR of the reciprocally methylated Nanog and Elf5 promoters in ES and TS cells. Note that both promoters are non-CGI promoters with <50% GC content and are efficiently pulled down and enriched in TS cells (Nanog) and in ES cells (Elf5). (B) Bioinformatic analysis of 2kb tiling probes covering the entire genome and showing that the vast majority of genomic fragments are of low-to-medium CpG content, which includes weak CpG islands that are predisposed to de novo methylation during differentiation, whereas fragments over 5% CpG represent a small minority of the genome. (C) Read count (=signal) distribution relative to CpG content in all of the 4 stem cell types analyzed by MeDIP-Seq. Importantly, the increase in signal between 0-3% CpG that would form the basis for a Batman correction is closely overlapping in all 4 stem cell types (i.e. corrected and uncorrected datasets are almost identical), therefore allowing a direct comparison of MeDIP signals between the stem cell types. The higher signal of high-CpG probes in particular in XEN cells is most likely due to their global hypomethylation (see Fig. 1B). For globally hypomethylated genomes, the remaining methylated fragments become seemingly enriched if the total number of sequenced reads is comparable between samples (see also Supporting Information Table S1). This overrepresentation due to global hypomethylation was corrected for in all analyses by normalization against total 5mC levels determined by mass spectrometry. (D) Graph showing the relative enrichment of different repeat elements for all the stem cell types displayed as percent of total reads. (E) Heat maps displaying Pearson's correlation of global methylation (5kb in silico probes spaced 20kb apart) vs CpG island methylation (CGI; as defined by Illingworth et al, 2010 [35]) between each stem cell type. Values were normalised for total read count and converted to a Log2 scale.
sc-12-0684_SupplFigure3.pdf559KFigure S3: (A) Scatter plots displaying Pearson's correlation of global methylation between the two cell lines of each stem cell type. Each point corresponds to a 5kb in silico “probe”. Each probe was spaced 20kb apart. Values were normalised for total read count and converted to a Log2 scale. (B) A neighbour joining tree based on a Pearson's correlation distance matrix of promoter methylation using bioinformatically defined promoter probes of −2kb − +100bp around the transcription start site. (C) Detailed analysis of promoter methylation differences in EpiSCs, TS and XEN cells compared to ES cells. Promoters were bioinformatically defined as −2kb − +100bp around the transcription start site, and percentage of 4-fold read count differences present in both cell lines of each stem cell type are shown. Promoters were classified into CpG island (CGI) promoters (Illingworth et al., 2010 [35]) and non-CGI promoters. Methylation at intergenic CGIs is also shown. The precise number of differentially methylated elements detected by this stringent cut-off method is given. (D) Venn diagram illustrating the overlap in promoters that are commonly hypermethylated, detected by 4-fold more reads, in both TS and XEN cells compared to ES cells. (E) Table showing the numbers of promoters commonly hypermethylated in EpiSC & TS, TS & XEN, XEN & EpiSC, and all three (EpiSC, TS & XEN) stem cell types compared to ES cells. (F) Gene ontology analysis of promoters that are commonly methylated (i.e. that have 4-fold more reads) in both TS and XEN cells compared to ES cells.
sc-12-0684_SupplFigure4.pdf873KFigure S4: (A) Confirmation by Sequenom analysis of differential methylation levels at various additional embryonic genes that were identified in the MeDIP-Seq approach. As expected all genes analyzed showed higher methylation levels in TS and XEN cells. (B) Tfap2c methylation determined by Sequenom analysis in the 4 different stem cell types corroborates promoter methylation in XEN cells.
sc-12-0684_SupplFigure5.pdf1115KFigure S5: (A) RT-qPCR analysis of embryonic genes Hoxb4, Hoxd13, Otx2, Pax6 and Nkx6.1 expression in wild-type E8.5 embryos and wild-type, Dnmt1 +/− and Dnmt1−/− trophoblast. Data are normalized against Sdha and Dynein and shown as mean ± SEM. No global re-activation of embryonic genes methylated in extraembryonic tissues is detected in Dnmt1−/− trophoblast tissue. E = embryos, T = trophoblast. (B) Screenshots of MeDIP-Seq datasets at key lineage transcription factors. The critical promoter region of the trophoblast “gatekeeper” gene Elf5 is only hypomethylated in TS cells (transcription start is indicated by the arrow; note that an annotated upstream exon is irrelevant for these stem cell types). ExEnd transcription factor Sox7 is methylated in TS cells, and conversely trophoblast transcription factor Eomes is highly methylated in XEN cells. Lack of methylation around the putative promoter regions of Pdzd3 and Klk1b11 specifically in XEN cells represent examples for the identification of potentially important lineage regulators through this MeDIP-Seq analysis. (C) Bisulphite sequencing analysis of the 5′-CGI region of Cdx2 in Reichert's membrane (RM) and ectoplacental cone (EPC) representative of the extraembryonic endoderm and trophoblast cell lineage, respectively. No methylation enrichment is observed in this region in RM, suggesting epigenetic repression by other epigenetic mechanisms such as H3K9me3 [43].
sc-12-0684_SupplTable1.pdf74KSupplementary Table 1
sc-12-0684_SupplTable2.pdf75KSupplementary Table 2
sc-12-0684_SupplTable3.pdf44KSupplementary Table 3
sc-12-0684_SupplTable4.pdf47KSupplementary Table 4
sc-12-0684_SupplData.pdf21KSupplementary Data

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