CyT49 (Viacyte proprietary male hESC line, normal karyotype) was grown as a monolayer culture by their standard methods [K. DÕAmour, personal communication]. Briefly, cells were thawed at 10 x 10^6 cells per T175 flask and grown in DMEM/F12 media (Gibco), 10% Xenofree Knockout Serum Replacement (Gibco), 1x non-essential amino acids (Gibco), 1% Glutamax (Gibco), 1% penicillin/streptomycin plus 10ng/ml activin A and Heregulin _1 at 37¡C and 8% CO2. Prior to initiating cellular differentiation, formation of cellular aggregates was achieved by placing the single cell suspension of undifferentiated ESCs into a rotational culture in 6-well, low attachment tissue culture plates in ESC growth media. Plates were rotated at 95 rpm overnight. For initiation of differentiation, the aggregates were pooled and washed with PBS prior to resuspending in day 0 differentiation media. Cells were treated with ActivinA + Wnt3a for one day, ActivinA alone for one day, KGF+ITS+TGF-Inhibitor for 3 days, Cyclopamine+TTNPB+Noggin for 3 days, KGF+EGF+Noggin for 3 days. Samples were taken at days 0, 1, 2, 5, 8 and 11 10 x 10^6 d0 cells and 3000 aggregates (d1-11) were fixed in 11% formaldehyde for ChIP-seq as described http://www.genpathway.com/support/fixation.html.