Sequencing And Alternative Splicing Analysis Of Single Cell Transcriptome Traced By CD133 Pedigree In The Fourth Ventricle Of Mice

The complexity of brain cell types is well known.Only by thoroughly understanding the interaction of various components and cell types in central nervous system tissues can we better understand the physiological function and pathological state of the central nervous system.The existing cell classification methods limit the resolution of cell types in the selection of markers and are difficult to reflect the heterogeneity of the central nervous system.Single cell transcriptome sequencing technology enables tissue heterogeneity to be reflected to the greatest extent by obtaining single cells without bias.Neural stem cells have the ability to differentiate into various types of central nervous system neurons and glial cells.In the past ten years,researches on adult neurogenesis have mainly focused on two brain regions,namely,the subventricular region(SVZ)of the lateral ventricle and the subgranular region(SGZ)of the hippocampal formation.However,there are few related studies on the fourth ventricle.In the previous work of our laboratory,we found that there are resting neural stem cells that can be activated by VEGF cytokines in the fourth ventricle.At the same time,existing research results show that alternative splicing plays a key role in neuronal development and mature neuronal function.In this study,we traced mice by constructing CD 133 lineage and sequenced the Smart-seq2 single cell transcriptome of the cells in the fourth ventricle.A total of 96 ZSGreen positive single cells were captured.unsupervised cluster analysis of the sequencing results showed that CD133+cells in the fourth ventricle can differentiate into many cell types under physiological conditions,such as neurons,ependymal cells,mature oligodendrocytes,microglial cells,endothelial cells,myelinated oligodendrocytes,etc.myelinated oligodendrocytes can also be divided into two subtypes.Through pseudo-timing analysis of sequencing results,the system believes that endothelial cells and ependymal cells are in the early stage of differentiation,while neurons,microglia and myelinated oligodendrocytes are in the late stage of differentiation.Five common alternative splicing types of TSS,TTS,IR,AE and SKIP5 were detected in this sequencing,and TSS and TTS accounted for the highest proportion and IR for the lowest proportion in all cell types.At the same time,this sequencing found that there was strong heterogeneity in the alternative splicing of the cells in the fourth ventricle of mice,which showed that there were many alternative splicing in the same cell.Different alternative splicing forms exist for the same gene in different cells of the same type.Through ORF prediction and amino acid sequence prediction of new alternative spliceosomes,it is found that some new alternative spliceosomes still have protein coding capability,while some new alternative spliceosomes no longer have protein coding capability.