Activin-A stimulation of ES cells was performed as follows. ES cell lines were started on gelatinized plates in ESC Growth Media. Cultures were then grown for 2 days in ESC Growth Media without LIF. On the third day, cells were cultured in low serum ESC Growth Media without LIF (same as ESC Growth Media except 0.1% FCS is used instead of 15% FCS) in the presence or absence of 30 ng/ml activin-A (R & D Systems) overnight. Following activin-A induction cells were washed with PBS and fixed in 2 mM EGS (Pierce) in 25% DMSO/75% PBS for 30 min followed by 1% PFA in PBS for 30 min. For histone ChIP, cells were fixed in 1% PFA in PBS for 15 min. Following fixation cells were washed 3X in PBS and removed from the tissue culture dish with a cell scraper. Cell pellets were then frozen at −80°C. The following day the pellets were thawed and processed using the ChIP procedure published by Upstate biologicals. Antibodies used for pulldown were ChIP grade SNF2H/L (Abcam), H3 3me-K4, H3 3me-K27 (Upstate) and pan H3 (Abcam).
