We characterized the neural identity and presence of molecular heterogeneity in our NGN2-iN culture using immunofluorescence of TUBB3 and PRPH (Figures S1A and S1B). The percentage of PRPH+ cells was quantified to compose 67% of neural cells, comparable with the percentage estimated by scRNA-seq (Figures 1E and S1B). We examined the presence of common makers that were used to characterize NGN2-iNs and how they overlap with PRPH expression (Figures S1C–S1E). Most NGN2-iNs and PRPH+ cells express CUX1 and VGlut1, but not GAD1/2, supporting their cortical excitatory feature as reported previously (Zhang et al., 2013). However, unlike PRPH and other identified cluster markers, common neural markers are not able to resolve the heterogeneity in our dataset. We noticed that the percentage of PRPH+ cells in our dataset is higher than previous reports (Chen et al., 2020; Nickolls et al., 2020; Schörnig et al., 2021). This can be due to differences in protocol, the particular readout of neural identity used in the previous reports, or thresholds for assigning positive staining from immunohistochemistry (Figure S2). Together, these data suggest that NGN2-iNs generated from
