E14 mouse embryonic stem cells (mESCs) were cultured with mouse ESC medium (DMEM high glucose supplemented with 15% ES FBS (Gibco), 2mM L-glutamine (Gibco), 1X PenStrep (Gibco), 100μM MEM non-essential amino acids (Gibco), 100μM β-mercaptoethanol (Gibco), 1000 U/mL leukemia inhibitory factor (LIF; ESGRO, Millipore)),  3 μM CHIR99021 (Tocris) and 1 μM PD0325901 (Tocris). NrESC medium is composed of ESC medium supplemented with 10 µM T0901317. All the ESC lines used in this study were cultured on gelatin-coated tissue culture plates and all the cell lines were cultured at 37°C with 5% CO 2 . Total RNA was extracted from each sample using Monarch® Total RNA Miniprep Kit (New England Biolabs, T2010S). Isolated RNA was then used for RNA-seq library preparation using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, E7775L), following manufacturer's instructions.