| In recent years,the spatial transcriptome sequencing technology with single-cell resolution has been rapidly developed,which helps to analyze the transcriptome information of a single-cell at a specific spatial location in tissue samples,and brings dawn to the drawing of cell maps and the development of brain science.The spatial transcriptome sequencing technology with single-cell resolution has already started,and there are still many problems to be solved.Among them,the steps of sample preparation are complex and influenced by many factors,and there is no stable plan yet.The processing of tissue samples,the preparation of tissue sections,the lysis of single-cell samples,and the construction of micro-sample sequencing library are all key factors that determine the quality of spatial transcriptome sequencing data.In view of these facts,we systematically investigated in this study 1)sample protection protocols for maintaining high-quality RNA of frozen tissue sections after room temperature treatment;2)obtaining microsample library preparation protocol for high-quality spatial transcriptome data;3)In order to further verify the effect of the sequencing sample preparation scheme,we also established a mouse model of Parkinson’s disease in this study,and performed transcriptome sequencing and data analysis of single-cell micro-region samples from different brain regions and different spatial locations,which laid a foundation for the in-depth research of the pathogenesis of Parkinson’s disease and the precise medical treatment of the disease.The research contents are as follows:1.Research of sample preparation for spatial transcriptome sequencing(1)To explore the effects of storage temperature,storage time and staining on the RNA integrity and gene expression of tissue frozen sections.The results showed that the RNA integrity number(RIN)of tissue sections decreased significantly with the extension of storage time at room temperature,and it was lower than 3 after 8 h.At-20°C,the RIN value of RNA in tissue sections didn’t decrease significantly with the increasing of storage time.High-throughput RNA-Seq results showed that the number and expression level of differential genes between samples at different storage times were closely related to the RIN value,so the RIN value could be used as an indicator to evaluate whether tissue section RNA could be used for spatial transcriptome sequencing.Tissue frozen section staining is an important means to observe cell morphology.The results showed that using cresol violet solution configured with 70% ethanol for staining,the integrity of the tissue section RNA higher,more suitable for spatial transcriptome sequencing research.Based on the above research results,the lower the temperature and the shorter the storage time,frozen tissue sections have higher RNA quality,and the use of 1% cresyl violet solution prepared with 70%ethanol to stain cells in sections can obtain better RNA integrity.(2)Research on methods to maintain high-quality RNA in frozen tissue sections at room temperature.Conventional frozen tissue sections are difficult to maintain RNA integrity at room temperature,and can’t meet the needs of single-cell micro-region sampling for transcriptome sequencing.In this study,the slice RNA stabilized solution(SRSS)was established.Brain tissue samples treated with PBS perfusion were soaked in SRSS solution to evaluate the RNA integrity and transcriptome sequencing data quality of frozen tissue section samples after treatment.The results showed that the tissue sections treated with SRSS could be stored stably for 24 h at room temperature,which fully met the requirements of single-cell sampling.The sequencing results showed that the quality of transcriptome data,the number of identified genes,the proportion of marker genes,and the accuracy of cell classification in the frozen tissue treated with SRSS solution were significantly higher than those of the control sample.(3)Research on the preparation protocol of micro sample transcriptome sequencing library.To improve the success of single-cell micro-region sample library construction and the quality of sequencing data,we optimize the reverse transcriptase,template switching oligonucleotides(TSO)and RNA samples structure based on the template transfer library construction strategy in this study.We established an ultra-low input RNA sequencing strategy(ul RNA-seq)based on Maxima H reverse transcriptase,universal TSO sequence(r Nr G+G)and sample RNA capping(m7G)processing,and obtained 0.5 pg RNA input amount of high-quality transcriptome data,improved the detection ability of low-abundance RNA,laid the foundation for single-cell level spatial transcriptome sequencing.2.Research on spatial transcriptome sequencing of mouse model of Parkinson’s disease(1)Transcriptome sequencing of different brain regions in mouse model of Parkinson’s disease.In this study,based on previous experiments,the transcriptome sequencing of samples from different brain regions(cerebral cortex,hippocampus,striatum and cerebellum)of the Parkinson’s mouse model was carried out.The results showed that compared with the control group,more differentially expressed genes were detected in the hippocampus and striatum of Parkinson’s mice,among which Lrrk2,Drd2,Ppp2 ca,Mtor,Adcy1,Gnai1 and Gnai3 had the largest differential expression changes.Differentially expressed genes between the striatum and hippocampus of Parkinson’s disease model mice were mainly involved in posttranscriptional regulation of gene expression and postsynapse,which involved AMPK,PI3K-Akt signaling pathway and GABA-ergic synaptic.It revealed that hippocampus and striatum play a more key role in the occurrence and development of Parkinson’s disease.(2)Research on spatial transcriptome of single-cell resolution sequencing in the hippocampus of mouse model of Parkinson’s disease.In order to verify the feasibility of the established frozen tissue section protection protocol and micro-sample library construction method,glass microneedles(20-30 μm diameter)were used to collect single-cell micro-samples from different spatial locations in the mouse hippocampus,and spatial transcriptome sequencing was performed.The results showed that the cell types in the hippocampus of Parkinson’s mice were mainly excitatory neuron,and there were great differences in gene expression patterns in different locations.The up-regulated differentially expressed genes Tubb2 a,Eno1,Atp2b1,Plk2,Map4,Pex5 l,Fibcd1 and Pdzd2 were mainly It is involved in neuron to neuron synapse,vesicle-mediated transport in synapse,Calcium signaling pathway and neurodegenerative disease pathways,while the down-regulated differentially expressed genes Sh3gl2,Aldoa,Stxbp6 and Camk2 g are mainly involved in ATP metabolism and Gn RH signaling pathway.The key genes identified in this study are expected to become new targets for disease treatment,provided new ideas for in-depth exploration of the pathogenesis of Parkinson’s disease.In conclusion,we established a set of brain frozen tissue section sample protection scheme and micro-sample transcriptome library strategy that can meet the requirements of spatial transcriptome sequencing,and obtained high-quality single-cell micro-region sample transcriptome sequencing data.In order to further verify the feasibility of the sample preparation scheme for spatial transcriptome sequencing,we took Parkinson’s disease model mice as an example to conduct transcriptome sequencing analysis of single-cell micro-region samples from different brain regions and different spatial locations,revealing the key brain regions and important target genes in the hippocampus of Parkinson’s disease,and providing a new idea for the mechanism research and targeted treatment of Parkinson’s disease. |
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