We manipulated NGN2 expression by shortening the duration of Dox treatment and analyzed the resulting cells at d14 using scRNA-seq (Figure 4A). A total of 2,767 cells (d1, 311 cells; d3, 378 cells; d5, 727 cells; d14, 1,351 cells) were included in the analysis. As expected, the expression level of exogenous NGN2 is correlated with the duration of Dox treatment (Figure 4B). Endogenous NGN2 expression is also positively correlated with Dox treatment duration, likely as a result of positive autoregulation (Figure 4B) (Ejarque et al., 2013). Each of the previously identified major clusters were detected in all samples; however, the duration of Dox treatment affects the proportion of samples among each cluster (Figures 4C–4E). Specifically, we found that the GPM6A+ population was enriched in samples with shorter Dox treatment, while the PRPH+/POU4F1+ population was more abundant in samples with increased Dox treatment (Figure 4E). Given the observation that NGN2 expression could affect NGN2-iN fate specification, we revisited the time course data for NGN2-iN development. The expression of NGN2 remained nearly consistent during NGN2-iN development, with a slight increase of NGN2
