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Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 35 cell properties, including depolarized resting membrane potential, lower input resistance, smaller maximum sodium currents and smaller after-hyperpolarization (Fig S1E-H). Spontaneous AP firing or incoming spontaneous synaptic responses were only observed in a few neurons (4/22). This is in line with previous studies reporting that synapse formation typically starts around two weeks after neuronal induction in human iPSC-derived neurons (Zhang et al. 2013). Together, the immunofluorescence and electrophysiology data indicate that human iPSC-derived neurons develop functional axons after neuronal polarization. Transcriptomic profiling of developing human iPSC-derived neurons To assess global changes in gene expression during differentiation and neuronal polarization, we next performed an unbiased, in-depth analysis of the transcript expression profiles during early neuronal development. To this end, we collected human iPSC-derived neurons at the previously described developmental stages andmonitoredmRNAexpression changes using quantitative population-based transcriptome analysis. The synchronized differentiation and relatively slow development of these cultures enabled us to select time points at which particular stages manifested in the majority of the cells. The cells were sampled for RNA analysis at days 1, 3, and 7, corresponding to stage 1, the onset of stage 2, and the onset of stage 3, respectively. At the same days, we collected samples for in- depth proteome analysis, which is discussed below. In two biological replicates with each two technical replicates, we identified transcripts corresponding to 14,551 genes by RNA sequencing (Table 1, Fig S2A). Of these, 9,655 transcripts were successfully quantified at all time points and normalized to reads per million for further analysis (Table 2). As expected, most genes with significantly altered expression were found between day 1 and day 7: 614 genes were downregulated and 549 genes were upregulated at day 7 compared to day 1 (FDR <0.05 (Yoav Benjamini and Yosef Hochberg, 1995)) (Table 3, Fig 2A, Fig S2B). Gene ontology (GO) enrichment analysis of downregulated genes indicates that many of the top enriched GO terms relate to processes involved in cell proliferation, such as DNA replication, cell cycle, and cell division (Table 4, Fig 2B). Upregulated genes correspond to several cellular components and biological processes including nervous system development, neuron projection, and axonogenesis (Table 4, Fig 2C). Consistently, upregulation of many neurodevelopmental-related and axonogenesis-related genes was previously observed in differentiating mouse embryonic stem cells (ESCs) and iPSCs as well as human ESCs (Wu et al. 2010; Chen et al. 2013). Interestingly, upregulated genes also showed enrichment of several GO terms related to the microtubule cytoskeleton (Fig 2C). Among the highly downregulated genes were several proliferation factors, such as SOX2, NOTCH1, and OTX2 (Table 3). Furthermore, the chromokinesin motor proteins KIF4a and KIF22, involved in cell proliferation by regulating spindle microtubule dynamics during mitosis, were downregulated (Almeida and Maiato 2018; Bisht, Tomschik, and Gatlin 2019). Conversely, genes of several neuronal and axonal kinesins are upregulated, including KIF1a, KIF5c, and KIF21b (Table 3) (Hirokawa and Tanaka 2015). Additionally, the neuron- specific tubulinTUBB3 and the axonal microtubule associated proteinMAPTare upregulated