Differentiation assays for dual reporter mouse embryonic stem cells (ESCs) were conducted as described previously [43 (link)], though compound treatment windows were modified as indicated. Flow cytometry was performed on a BD Accuri C6 or BD LSRFortessa flow cytometer. Synthesis of compounds used in the present study was performed in the Division of Pharmaceutical Chemistry at the University of Helsinki, Pharmatory (Oulu, Finland), Chembridge (San Diego, USA), and Maybridge (Leicestershire, UK) as described [38 (link), 39 (link)]. All-trans retinoic acid (ATRA) and (+)-JQ1 were purchased from a commercial provider (Sigma). Compounds were diluted in DMSO prior to administration (final DMSO concentration 0.1% in medium) and values were normalized to DMSO controls. For characterization of chemically differentiated embryoid bodies (EBs) by qRT-PCR, RNA was isolated from D12 EBs using TRIzol reagent (Thermo Fisher Scientific) and RNeasy MinElute Cleanup kit. qRT-PCR reactions were performed using Taqman gene expression assays (Supplementary Table 1 ), and values were normalized to a reference gene (Actb). qRT-PCR reactions were performed on a Fluidigim Biomark HD system.
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