1. Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing 
John Huntriss, Ph.D., Kathryn Woodfine, Ph.D., Joanna E. Huddleston, Ph.D., Adele Murrell, Ph.D., Anthony J. Rutherford, MBBS, FRCOG, Kay Elder, Ph.D., Amir Ali Khan, Ph.D., Karen Hemmings, Ph.D., Helen Picton, Ph.D. 
Fertility and Sterility 
Volume 95, Issue 8, Pages e8 (June 2011)
DOI: /j.fertnstert
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Methylation analysis of KvDMR1, RB1, and SNRPN by pyrosequencing in individual human blastocysts. All samples are described in Supplemental Table 1 (available online). Each data point represents methylation at a single CpG site within the DMR. The mean methylation for all sites in this DMR is shown for each sample (horizontal lines). (A) Methylation levels for KvDMR1 from blastocyst samples 1–8 and adult human kidney template DNA control (sample 9). (B) Methylation analysis of the RB1 gene by pyrosequencing in five individual human blastocysts (samples 12–15, 17). Sufficient DNA was available in three blastocysts to repeat the RB1 assay and experiments were designated “a” or “b” (samples 13a/13b, 14a/14b, 17a/17b). A pooled blastocyst sample (sample 18) and the blood template DNA control (sample 19a/19b) were included. (C) Methylation analysis of the SNRPN gene by pyrosequencing in five individual human blastocysts (samples 10–12, 15, 16). A pooled blastocyst sample (sample 18) and blood DNA template control are included (sample 19).
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3. Supplemental Figure 1
Data from Figure 1 plotted in series showing methylation at each CpG site across each imprinted gene DMR. (A) KvDMR1 methylation plot with hypermethylated (sample 8) and partially hypomethylated (sample 5) embryos. (B) RB1 methylation data. (C) SNRPN methylation data.
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Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

4. Supplemental Figure 2
Pyrograms from selected embryos for KvDMR1 (blastocysts samples 4 and 6), RB1, (blastocysts 14 and 17), and SNRPN (blastocysts 11 and 15). The pyrograms show quantitative methylation data at individual consecutive CpG sites across each DMR. Cytosines that are potentially methylated (either C or T after treatment with bisulfite) within the DMR are shown as gray bars, with the methylation level reported above.
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Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

5. Supplemental Figure 3
Methylation analysis by pyrosequencing of 10 imprinted gene DMRs in human embryonic stem cells and blood DNA. The assays were DIRAS3, ZAC1, GRB10, MEST, KvDMR1, RB1, MEG3, SNRPN, NESP55, and MCTS2. (A) The H1 human embryonic stem cell line, passage 44. (B) The HuES1 human embryonic stem cell line, passage 51. (C) Blood. Hypermethylation of RB1 (91.8% mean methylation for H1 and 75.6% for HuES1), and MEG3 (91.7% mean methylation for H1 and 83.5% for HuES1) was observed in both hESC lines. Hypomethylation at DIRAS3 was observed in HuES1 (14.8%). Methylation at the remaining loci in hESCs was between 38.5% and 56.7%, consistent with imprinting. Methylation in the blood DNA control that was run in the same experiment was DIRAS3 56.7% (3 CpGs), ZAC1 44.6% (7 CpGs), GRB % (14 CpGs), MEST 66.2% (6 CpGs), KvDMR1 52.6% (12 CpGs), RB1 51.8% (9 CpG sites), MEG3 48.8% (7 CpGs), SNRPN 49.2% (5 CpGs), NESP % (8 CpGs), MCTS2 44.7% (3 CpGs). The human embryonic stem cell lines were supplied by the School of Biology, University of Leeds and cultured on mitotically inactivated mouse embryonic fibroblasts feeder layers in 80% (vol/vol) knock-out Dulbecco’s modified minimal essential medium (DMEM; Invitrogen), 20% (vol/vol) Knock-Out serum replacement (Invitrogen), supplemented with 1% (vol/vol) ×100 nonessential amino acids, 1 mM l-glutamine, 0.1 mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions