Distinct DNA methylation patterns characterize differentiated.doc

SUPPLEMENTAL TEXT Supplemental Methods Tissue samples and nucleic acid preparation We obtained purified genomic DNA and total RNA from human liver tissue from Asterand (Detroit, MI). These adult tissues from Asterand were surgically excised during removal of a nearby tumor, but pathological examinations indicated normal tissue. Consented fetal liver samples were isolated from legally aborted fetuses at 11 and 24 weeks gestation. Total RNA and genomic DNA were prepared from these tissues by using Trizol (Invitrogen) and DNeasy kit (Qiagen), respectively. We assessed the purity of the RNA and DNA by using a NanoDrop device as well as by using agarose gel electrophoresis. Cell Culture HCT116 cell cultures were grown according to standard protocols (ATCC). Human embryonic stem cells (hESCs) H9 (XX euploid) and BGO2 (XY euploid) were obtained respectively from WiCell and Bresagen. Undifferentiated hESCs were maintained on mouse embryo fibroblast (MEF) feeder layers or on Matrigel (1:20 dilution; BD Biosciences) in mouse embryo fibroblast-conditioned medium (CM). CM was produced by conditioning of MEFs for at least 24 hours in Dulbeccos modified Eagles medium/Hams F-12 medium (DMEM/F12) supplemented with 20% knockout serum replacement (Gibco), 1 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, and 8 ng/ml recombinant human fibroblast growth factor-basic (bFGF; Peprotech). Cultures were routinely passaged with 200 U/ml type IV collagenase (Gibco) at the split ratio of 1:3 to 1:4 every 4–5 days. We karyotyped the cultured hESC lines and determined that they were euploid for this analysis and also made sure that the analysis was performed after a low number of passages (30-40). In vitro differentiation Definitive endoderm precursors were generated from hESCs as previously described (DAmour et al. 2005). Differentiation was performed in RPMI-1640 medium supplemented with glutamax, 100 ng/ml recombinant human activin A (RD Systems), penicil