Kapa taq kit

In vitro mRNA synthesis and transfections were performed according to previous protocols.^{46 (link)} In brief, T7 promoter and polyA tail were added by polymerase chain reaction (PCR) using KAPA taq kit (Kapabiosystems, London, UK). RNA was transcribed from the template using MEGAscript T7 kit (Ambion, Carlsbad, CA, USA), with ARCA cap analog (New England Biolabs, Ipswich, MA, USA); ATP; GTP; 5-Methyl-CTP (TriLink, San Diego, CA, USA); and pseudo-UTP (TriLink). Synthesized RNAs were purified with the MEGAclear kit (Ambion). RNA transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To increase the viability of transfected cells, B18R interferon inhibitor (eBioscience, San Diego, CA, USA) was supplemented to the culture medium. One day before transfection, 30,000 hESCs were seeded on a culture plate, and 1 µg/well of each synthetic modified mRNA was induced. The hESCs were subjected to four consecutive transfections with TF-encoding RNAs, or GFP or mCherry mRNAs as controls with Lipofectamine 2000 (Life Technologies) in StemFit AK-03 with B18R (eBioscience) for the first two days of differentiation. After two consecutive days of transfection, the culture medium was changed to DKSFM with 100 μg/mL CT and 10 ng/mL EGF.