To investigate the molecular events underlying the dramatic developmental changes, we identified 3,231 genes with significant expression changes along the course of NGN2-iN development and segregated them into six clusters with their expression peak at different stages (Figure S3E). We performed functional enrichment analysis on these differentially expressed (DE) genes and recovered gene ontology terms related to neural development (Figure S3F; Table S1). We cross-referenced DE genes with annotated transcription factors (TFs) (Hu et al., 2019) to identify potential drivers of gene expression changes during NGN2-iN development (Figure S1G). We focused on TFs that changed from 6 to 12 h to d1 after Dox induction and constructed a gene-regulatory network (Aibar et al., 2017), incorporating transcription factor binding site prediction in promoters with TF-target co-expression (Figure S3H). NGN2 was predicted to connect with some TFs with the highest centrality in the constructed regulatory network, including POU5F1, HES6, and SOX11, supporting its role in driving direct reprogramming from iPSCs to induced neurons.
