Previous studies suggested that iN cultures may be heterogeneous, potentially confounding a bulk RNAseq analysis [30]. To enable analysis of iNs, we used single-cell RNAseq (scRNAseq) of pooled neurons from multiple cell lines, a strategy known as a “cell village [37].” Based on preliminary results indicating differences in excitability, we cultured each haplotype separately to avoid potential secondary effects, combining cells within each group in equivalent proportions. Following maturation (~30 days after induction), dissociated cells were processed to generate scRNAseq libraries. Mapping cells by t-distributed stochastic neighbor embedding (tSNE), a distinct cluster of cells coordinately expressed several markers consistent with neuron physiological function (Fig. 1C and Supplementary Fig. 2E), including synaptophysin (SYP), voltage-gated sodium channel (SCN3A), glutamate transporter (SLC17A6), and both NMDA (GRIN2A, GRIN2B, GRIA2, GRIA4) and kainate (GRIK2) glutamate receptors. This cluster also expressed both KCNJ6, encoding GIRK2, and KCNJ3, encoding GIRK1, which are required to form heterotetrameric, functional channels [14, 38]. Detection of mRNA in individual cells by scRNAseq underestimates expression, so the cluster identified as neurons likely expresses markers more uniformly than observed here. We conclude that
