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. 2020 Jun 22;147(12):dev184093. doi: 10.1242/dev.184093

Fig. 1.

Fig. 1.

Endogenous tagging reveals E2A is expressed heterogeneously in pluripotent cells. (A) qRT-PCR analysis of pluripotent ESCs plated under standard neural differentiation conditions. Expression values are normalised to the housekeeping gene Sdha. Data are mean±s.d. n=3 biological replicates. (B) Schematic of wild-type (WT) and tagged E2A locus. The V5 epitope tag was knocked-in at the 3′ UTR of the endogenous E2A locus. The V5 tag is shown in green and the stop codon in red. (C) Western blot analysis of E2A-V5 cells during neural differentiation. β-Tubulin was analysed as a loading control. (D) Immunostaining of parental wild-type ESCs and epitope-tagged E2A-V5 ESCs. Cells co-stained for the V5 tag, the pluripotency marker Nanog and nuclear marker DAPI. (E) Immunostaining of E2A-V5 cells from days 2 to 5 of neural differentiation. Cells were stained for Oct4 and Sox2 to enable identification of cells committing to the neural lineage (Oct4−/Sox2+). Scale bars: 30 µm.