ES cell differentiation

Two of the male and female ES cell lines from which ChIP-seq was performed were subjected to a standardized differentiation protocol that directs the stepwise differentiation of early embryonic cells into cardiac precursors [41], as assayed by marker gene analysis. Cells were cultured with leukemia inhibitory factor (LIF) on mouse embryonic fibroblasts (MEFs). Before differentiation, ES cells were dissociated, MEFs were eliminated as detailed above, and embryoid bodies were derived by hanging drop culture in medium without LIF. After 4 days, embryoid bodies were harvested and grown in medium containing Activin A, BMP4, and VEGF as monolayers until beating foci were observed. This optimized protocol yields > 75% cardiomyocytes [41]. At day 13 of initial LIF withdrawal, we picked beating foci from the plates and obtained RNA.

qPCR was performed to determine expression of pluripotency markers Nanog and Oct4 and cardiomyocyte markers Myh6 and Tnnt2 on cDNA generated using SuperScript^TM II (Invitrogen) and relative expression was assessed using PowerUp SYBR Green Master Mix (Thermo Fisher) and normalized to β-actin on the Applied Biosystems StepOnePlus Real-Time PCR System. RNA-seq was performed as previously described [24].