a, Immunocytochemistry of iASCL1 (iA - left panel) and iNEUROG2 (iN - right panel) embryoid bodies (EBs) showing the expression of neuronal Tubb3 and V5-tagged Ascl1 or FLAG-tagged Neurog2 transgene expression over time, respectively (scale bar: 1 µm). 0h time point (before induction of TFs) is used as a control. Similar results were observed in at least n = 2 cell differentiations. b, Schematic representation of time-series (bulk) RNA-seq experiments (not to scale) showing the overlap of the genes up or downregulated early (12 h) or late (48 h) between iA and iN neurons. c, Gene Ontology (GO-terms) biological process terms enriched at the early and late shared upregulated genes between iA and iN neurons (PANTHER- Fisher’s Exact test: FDR-corrected p-value < 0.05) (iA 12 h n = 2; iN 12 h n = 2; iA 48 h n = 5; iN 48 h n = 2 independent cell differentiations). d, Bulk RNA-seq heatmaps showing the expression of subtype-specific markers in iA and iN neurons 48h after induction (EB t = 0 n = 5; iA 48 h n = 5; iN 48 h n = 2 independent cell differentiations). log2(fold change) values are plotted with respect to before induction (EB t = 0) in magenta-orange heatmap. The ratio of iN (48 h) over iA (48 h) is plotted on the blue-green heatmap.