5G6GR mouse ES cells: scRNA-seq protocol

5G6GR mouse ES cells were used for total RNA extraction and scRNA-seq. 5G6GR mouse ES cells were provided by Hitoshi Niwa (Laboratory for Stem Cell Biology, Institute of Molecular Embryology and Genetics at Kumamoto University). This cell line was constructed by randomly incorporating the linearized Gata6-GR-IRES-Puro vector into EB5 ES cells^{46 (link)}. The cells were cultured in a feeder-free gelatin-coated dish in 10% fetal calf serum containing Glasgow minimal essential medium (Sigma-Aldrich), 1000 U/mL leukemia inhibitory factor (ESGRO; Millipore), 100 µmol/L 2-mercaptoethanol (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), 1 mmol/L sodium pyruvate (Thermo Fisher), 2 mmol/L l-glutamine (Sigma-Aldrich), 0.5× penicillin/streptomycin (Thermo Fisher), 0.5 µg/mL puromycin (Sigma-Aldrich), and 10 µg/mL blasticidin (Thermo Fisher). To assess PrE differentiation, 5G6GR ES cells were cultured in differentiation medium containing 100 mmol/L dexamethasone rather than blasticidin. The cells were cultured for 72 h.