6800 Nucleic Acids Research, 2007, Vol. 35, No. 20 values of the ﬁrst derivative of a cubic spline ﬁt. Plots of n against the number of CGs (n) exhibited a nearly linear relationship. Mapping the CG-dense fragments in Cn back to the genomic sequence produces an annotation track where each annotated locus is a conglomeration of one or more overlapping fragments of variable length. However, the exact number, length and location of the annotated regions vary with the number of CGs per fragment (n). As the basis for choosing the optimal track in an objective manner, we noted that the fragments tended to aggregate and overlap to a greater extent in genomic regions of higher CG density. Because these types of regions are the major source of the CG-dense subpopulation, we used the number of overlapping fragments at locus j, ðjOjnjÞ, as a parameter for evaluating the information content of an annotated locus. To normalize for the length dependence of this value, we divided it by the maximum fragment length n. To choose the track with maximal fragment overlap per locus, we compared genomic averages of this metric ðPj jOjnj= nÞ for diﬀerent numbers of CGs per fragment (n). This allowed us to choose the species-speciﬁc optimal number of CGs per fragment for the ﬁnal annotation. These annotations were then formatted for visualization in the UCSC genome browser and are available for download (human and mouse genomes) at http://greallylab.aecom.yu.edu/cgClusters/ Annotation track features including CpG islands and repetitive elements were examined using a local mirror of the UCSC genome browser MySQL database through the PERL DBI interface. The Takai and Jones (8) and Gardiner-Garden and Frommer (2) CpG island annota- tion tracks were generated using the cpgi130 program (8) (http://cpgislands.usc.edu/), and loaded into the database to facilitate analysis. The CG cluster annotation was also loaded into the database. Analysis of CpG island and CG cluster promoter prediction was performed using a highly restrictive set of criteria. Only refSeq genes were considered, and promoter prediction was deﬁned as strict overlap of the transcrip- tion start site. Non-transposon CG clusters were deﬁned by quantifying the number of CG dinucleotides derived from transposon and unique sequences, identifying those for which unique sequence contributed less than the minimum number of CGs required for a CG cluster in each species and removing them from consideration. For the comparisons of CpG islands and CG clusters at orthologous promoters in human and mouse at the 23 loci, we used the same approach as in the original analysis (15), scoring conservation when the promoter of the gene had any overlap with the sequence feature. For the corresponding genome-wide analysis of CpG island and CG cluster conservation, we deﬁned orthologous annotations in human and mouse using the mouse net (netMm7) track from the UCSC Genome Browser (16). Promoter hits were deﬁned as strict overlap with transcription start sites of refSeq genes, while overlap of the annotation from one species with the annotation in the other species at orthologous sequences deﬁned conservation of the CG-dense region. Cytosine methylation analysis using the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay Two normal human cell types were chosen for analysis, human embryonic stem cells and hematopoietic stem and progenitor cells. The H1 human embryonic stem cells (hESCs; NIH code WA01 from Wicell Research Institute, Madison, WI, USA) were cultured on P51R [hESC- derived MSCs (17)], plated at 75 000 cells per cm2 or on matrigel (BD Biosciences, San Diego) at 378C, 5% O2 and 5% CO2. The hESC medium contained DMEM/Ham’s F-12, 20% Knockout Serum Replacer (KSR), 2 mM L-glutamine, minimal essential medium nonessential amino acid solution (NEAA), 0.1 mM penicillin– streptomycin 1% (all from Gibco, Grand Island, NY, USA), 4 ng/ml basic ﬁbroblast growth factor (or 100 ng/ml for cells on matrigel, R&D Systems Inc., Minneapolis; or ProSpect-Tany, Technogene, Rehovot, Israel) and 0.1 mM 1-thioglycerol (Sigma–Aldrich, St Louis). The culture medium was changed daily, and the cells were passaged once weekly. The hESCs were harvested using TrypLETM EXPRESS (Gibco), washed and re-suspended in staining buﬀer [Dulbecco’s phosphate-buﬀered saline (DPBS) + 5% KSR] at a concentration of 107 cells/ml and stained with mouse anti-human SSEA-4 antibody (DHSB) or isotype control (eBioscience, San Diego). Secondary staining was performed using rat anti-mouse IgG (H+L) immu- noglobulin conjugated to ﬂuorescein isothiocyanate (eBioscience). Based on ﬂuorescence, positive cells for SSEA-4 were sorted using Moﬂow Cell-Sorter (DakoCytomation, Glostrup, Denmark). Genomic DNA was extracted from 1.5 to 2.5 106 cells using proteinase K digestion, phenol–chloroform extraction, dialysis against 0.2 SSC and concentration by surrounding the dialysis bag with PEG 20 000 to reduce water content by osmosis. CD34+ cells were selected from bone marrow samples of healthy adult donors using a Miltyeni (Auburn, CA, USA) LS immunoabsorption column. Genomic DNA was extracted from 2 to 3 106 cells following a standard phenol–chloroform protocol followed by an ethanol precipitation and re-suspension of the DNA pellet in 10 mM Tris pH 8.0. To identify hypomethylated loci, HELP analysis was performed (12) using a custom microarray representing HpaII-ampliﬁable sites at gene promoters (NimbleGen Systems). We used a categorical approach for the output of the assay, as our outcome of interest was deﬁned in terms of methylated or hypomethylated loci. Methylated loci were identiﬁed by their inability to amplify from HpaII representations of genomic DNA (measured by the median microarray ﬂuorescence intensities for the oligonucleotides representing each HpaII-ampliﬁable fragment, when median HpaII signal intensity was below the level of background signal, deﬁned as 2.5 median absolute deviations above the median of random probe signal intensities), despite ampliﬁcation in the corresponding MspI representation (signal intensity above the background calculated in the same way for the MspI channel).