Histone H3 (K9) methyltransferase activity assay

Nuclear extracts were prepared from undifferentiated Strip2⁻-ESCs, 4-day EBs differentiated and 16-day EBs as well as from the appropriate control Strip2⁺-ESCs and the 4-day and 16-day-EBs using Nuclear Extraction Kit (ab113473; Abcam, Cambridge, UK) by following the manufacturer’s protocols. The histone methyltransferase activity of the nuclear fractions of differentiating Strip2⁻-ESCs and differentiating Strip2⁺-ESCs were determined using the histone methyltransferase H3 (K9) activity quantification assay kit (ab113453; Abcam) following the manufacturer’s protocols. Histone H3 acetylation was determined by a histone H3 acetylation assay kit (ab115102; Abcam). The absorbance at 450 nm was measured using a plate reader. Methyltransferase activity was calculated by the formula: activity [OD/h/mg] = (Sample OD – blank OD)/(protein amount [μg] × incubation hour). Histone acetylation activity was calculated by formula: [ng/mg protein] = (Sample OD – blank OD)/(Slope) × 1000.