| In recent years,the emergence and development of single cell RNA sequencing(scRNA-seq)technology,especially the advent of high-throughput parallel scRNA-seq technologies such as Indrop,Drop-seq and Cyto-seq,enabled researchers to discover and evaluate the differential gene expression among different cell types in individual organisms,and reveal the transcriptome heterogeneity existing in biological tissues with single-cell resolution,so as to understand the growth and development process of individual organisms and the mechanism of disease occurrence.However,scRNA-seq requires the isolation of cell populations within tissues into single-cell suspensions,resulting in the loss of spatial location information critical to understanding cell development and gene expression dynamics.But in biological tissues with a large number of cell populations,the transcriptome and functions of a single cell are not only determined by its own differentiation fate,but also strongly influenced by cell-to-cell interactions through cell communication and its structural microenvironment.Therefore,the spatial location information of individual cells is of great significance.Spatial transcriptome technology can qualitatively and quantitatively analyze the transcriptome while retaining the original spatial location information.It can analyze the interaction mode between cells with different gene expression profiles and how microenvironment shaping cell transcriptome heterogeneity.An important step in the scRNA-seq and next Generation sequencing(NGS)-based spatial transcriptome methods is to convert m RNA from single cell or microregion tissues into c DNA libraries that meet the requirements of NGS sequencing.The quality of c DNA library construction method has a decisive influence on the transcriptome detection flux and effect,the amount of experimental operation and the cost of reagents.In this paper,we study the synthesis of Split-Combinatorial DNA Barcode(SCDB)microbeads and design a highly multiplexed medium-throughput(50-1000 samples)transcriptomic sequencing c DNA library construction method based on SCDB encoded microbeads.The main research work of this paper is as follows:1.Synthesis and optimization of SCDB microbeads: A unique synthesis scheme of DNA barcodes microbeads based on Split-Combinatorial concept was designed.96 kinds of DNA barcodes microbeads with known DNA barcodes sequences and corresponding relationship with micropore positions could be synthesized by using 20 coding primers.Compared with the traditional split-pool synthesized DNA barcodes microbeads,SCDB microbeads do not need to decode the DNA barcodes on the microbeads in some way,so it shows a wide application prospect in spatial transcriptomic sequencing.2.Construction of spatial transcriptome c DNA sequencing Library based on SCDB microbeads by modifying the Smart-3SEQ method to a certain extent,replacing oligo-d T with SCDB microbeads and improving subsequent corresponding experimental procedures to improve the quality of DNA sequencing library,a highly multiplexed medium-throughput(50-1000 samples)transcriptome sequencing method: SCDB-seq,which in combination with microdissection or microregion sampling,enables spatially-resolved transcriptome sequencing with low laboratory operation and low reagent cost.3.Spatial transcriptome analysis of mouse brain sections based on SCDB-seq: applying SCDB-seq to fresh frozen tissue slices and FFPE tissue slices of mouse brains to explore whether SCDB-seq could effectively analyze the spatial transcriptome of actual samples with complete or degraded m RNA molecules.According to the experimental results,SCDB-seq can selectively perform single-cell resolution and multiplexed spatial resolved transcriptome analysis on freshly frozen tissue sections and FFPE tissue sections,thus is capable fo analyzing gene expression in specific tissue microregions,providing insights for biological research,clinical diagnosis and diagnosis. |
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