Spray the abdominal fur with 70% alcohol. Sacrifice the pregnant mice by CO 2 administration. Here, we propose a method for the isolation of fluorescence-labeled ОІ cells from the pancreases of fetal to adult Ins1-RFP transgenic mice 11, using fluorescence-activated cell sorting (FACS), and the performance of transcriptomic analyses at the single-cell level, using Smart-seq2 technology ( Figure 1).This protocol can be extended to analyze the transcriptomes of all pancreatic endocrine cell types in normal, pathological and aging states.Д®stimate embryonic day 0.5 based on the time point when the vaginal plug appears. Smart-seq2 takes approximately two days to construct a cDNA library for sequencing 10. Among the single-cell RNA-sequencing protocols, Smart-seq2 allows the generation of full-length cDNA with improved sensitivity and accuracy, and the use of standard reagents at lower cost 9.

Taking advantage of this technology, the key events that occur during pancreatic islet development can be deciphered at the single-cell level 8. Single-cell RNA-sequencing is a powerful approach that can profile cell subpopulations and trace cell lineage developmental pathways 7. However, the regulatory mechanisms of these processes have not been well studied. Moreover, the ОІ cell maturation process also involves the regulation of cell number expansion 3, 4 and the generation of cellular heterogeneity 5, 6. These immature cells continue to develop and gradually become mature islets, which become richly vascularized to regulate blood glucose homeostasis in adults 2.ДЄlthough a group of transcriptional factors has been identified that regulate ОІ cell differentiation, the precise maturation pathway of ОІ cells is still unclear. Later, during the perinatal period, the endocrine cells proliferate to form immature islets. Pancreatic endocrine cells are derived from Ngn3 + progenitors during embryogenesis 1. Dysfunction of the endocrine cells results in diabetes mellitus, which has become a major public health issue worldwide. Pancreatic endocrine cells, including insulin-producing ОІ cells and glucagon-producing О± cells, cluster together in the islets of Langerhans and coordinately regulate systemic glucose homeostasis. The pancreas is comprised of endocrine and exocrine compartments. The pancreas is a vital metabolic organ in mammals.

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