p300, CBP siRNA knockdowns were preformed as follows. ES cells were grown in ES Cell Growth Medium with 100 U/ml LIF on gelatinized plates. Cells were collected using trypsin and transfected using an Amaxa Nucleofector device using solution ES Cell and program A-30 according to manufacturer's procedures. 2.0×106 ES cells were transfected with 400 nmoles siRNA duplex (Darmacon) or a GFP-Max nucleofection control. Post nucleofection 1.0×106 cells were plated to each well of a 6 well gelatinized tissue culture plate. Cells were stimulated with or without Activin-A in low serum ES Cell growth media as described above. Cells at specified time points were lysed using TriReagent reagent (Sigma) and RNA and protein were purified according to manufacturer's procedures.
