BioSample

Protocols: PSCs were cultured on maintenance media which was different for each species, including mouse (Mus musculus), marmoset (Callithrix jacchus), rabbit (Oryctolagus cuniculus), human (Homo sapiens), cattle (Bos taurus) and southern white rhinoceros (Ceratotherium simum). RNA was extracted on the day prior to splitting. To differentiate PSCs from different mammalian species into PSM-like cells, PSC cells were directly changed from the maintenance media to the differentiation media. The differentiation protocol consisted on culturing the cells from 2 to 3 days (depending on the species) in CDMi media containing Chiron (CHIR), bFGF, DMH1 and SB-431542. Human and Marmoset cells were first cultured for 24 hours in CDMi media with CHIR, bFGF and Activin A. Mouse cells were first cultured on their maintenance media without IWR-1 for 24 hours. RNA was extracted under the same culturing conditions during the most efficient day of differentiation based on the percentage of cells expressing the PSM marker TBX6. RNA samples were extracted from cultured cells using the RNeasy Mini Kit (cat. no. 74104) following the manufacturer's instructions. On column DNase digestion was performed on all samples. Barcoded stranded mRNA-seq libraries were prepared from 300ng of high quality total RNA samples using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs (NEB), Ipswich, MA, USA) implemented on the liquid handling robot Beckman i7.