In order to determine whether the hypermethylation and hypomethylation we observed at imprinted loci resulted in loss of imprinting, we examined allele-specific gene expression at a subset of imprinted loci. We first used SNP genotyping data we had previously obtained on our samples using the HumanOmni1 SNP genotyping microarray (Laurent et al., 2011) to identify which of the samples contained informative heterozygous SNPs in the PEG10 and PEG3 mRNAs. We then performed allele-specific real time polymerase chain reaction (RT-PCR) to show that loss of DNA methylation at the PEG10 locus correlated with biallelic expression in the ESI051p37 hPSC sample and that hypermethylation of PEG3 led to a total loss of gene expression in several hPSC lines in comparison to the monoallelic expression observed in parental fibroblasts and an adult bladder sample (Figure 4C–D). Using the HT12V3 mRNA expression array, we also determined that CpG methylation and mRNA expression were anti-correlated for MEG3, PEG3/ZIM2, NAP1L5, NNAT, GNAS, NDN, H19 and SNRPN (Table S5A). For many imprinted genes, our DNA methylation data show similar frequencies of either stable or aberrant CpG methylation
