To analyze the transcriptome of iNs via RNA-seq, neuronal RNA needs to be pure, with minimal fibroblast contamination that might confound analysis regarding cell aging. As live iNs express PSA-NCAM on the cell surface, we established a fluorescence-activated cell sorting (FACS)-based protocol for purification of neurons (Figures 3A and 3B). Following sorting and plating on astrocytes, over 90% of human cells expressed βIII-tubulin, with over 95% of them also being hTau-positive (Figure 3C). Following prolonged culture, almost all human cells showed mature neuronal morphologies and marker expression (Figure 3D). We next performed RNA-seq analyses over the time course of conversion and observed dramatic gradual changes in gene expression (Figure 3E). Neuronal genes such as Dcx, NeuroD2, and Mapt, and voltage-gated Na+ and K+ channels, synaptic proteins, and neurotransmitter receptors were strongly upregulated, whereas fibroblast genes such as dermokine (Dmkn), collagen, keratin, and cell-cycle genes such as Ccna2, Ccnb2, Nuf2, and Anln were dramatically downregulated (Figure S2; Table S4). Neighboring time points clustered together, indicating a gradual process of cell type conversion. Gene ontology (GO) term analysis of the 200 most
