Dynamic Transcriptomes during Neural Differentiation of Human Embryonic Stem Cells Revealed by Short, Long, and Paired-end Sequencing Running Title: RNA-Seq during Neural Differentiation of Human Embryonic Stem Cells

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of human embryonic stem cells (hESCs) into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation, N1 (initiation), N2 (neural progenitor cell), and N3 (expressed glial markers) were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call " isoform specialization. " During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal new insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, NPC identity maintenance and the transition from a predominantly neuronal state into one with increased gliogenic potential. 4 INTRODUCTION Neural commitment and subsequent differentiation is a complex process. Although the complexity of RNAs expressed in neural tissues is very high (1, 2), a comprehensive analysis of the genes and RNA isoforms that are expressed during the different stages of neural cell differentiation is largely lacking. Such information is expected to be important for understanding mechanisms of neural cell differentiation and ultimately providing therapeutic solutions for neural degenerative diseases, such as Parkinson's and Alzheimer's disease. Our current knowledge of the mechanisms involved in neural cell formation is derived mostly from studying neurogenesis in the developing embryos of animal models (3, 4). However, neurogenesis in animals is a complex process involving many different cell types that differentiate asynchronously. This heterogeneity, along with the relatively small number of cells that can be readily obtained, makes the analysis of the temporal differentiation of individual cell types extremely difficult. One solution is to analyze hESCs during in vitro differentiation to different stages of neural development, which can be performed using a relatively large numbers of cells (5–9). Analysis of the transcriptome in these cells is expected to provide insights into the mechanisms and pathways involved in early cell fate specification, such as the acquisition of neurogenic potential and the transition …