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CN-116334195-B - Space single cell transcriptome sequencing method based on light-controlled cell markers

CN116334195BCN 116334195 BCN116334195 BCN 116334195BCN-116334195-B

Abstract

The invention discloses a space single-cell transcriptome sequencing method based on light-controlled cell markers, which uses ONPF-biotin as a probe, and comprises the steps of marking cells to be sequenced by using ONPF-biotin, and sorting and sequencing the cells marked by ONPF-biotin. The invention realizes efficient light-operated cell marking and space single cell transcriptome sequencing, solves the problems of low marking efficiency, less marking position number, high background signal, dependence on genetic operation and the like of the traditional method, and provides a new generation solution for researching the space distribution and regulation of cells in different biological systems.

Inventors

  • CHEN XING
  • TANG QI
  • LIU LU
  • ZHANG XU
  • GUO YILAN
  • ZHANG SHAORAN

Assignees

  • 北京大学

Dates

Publication Date
20260505
Application Date
20211222

Claims (8)

  1. Application of ONPF-biotin as a probe to fluorescent labeling of cells at specific spatial positions in a spatial single cell transcriptome sequencing technology, wherein the ONPF-biotin has the structural formula: 。
  2. 2. A probe for spatial single cell transcriptome sequencing, wherein the probe is ONPF-biotin and the ONPF-biotin has the structural formula: 。
  3. 3. A method for preparing a probe according to claim 2, comprising the step of adding AzONPF and DBCO-biotin to a mixed solution of dimethyl sulfoxide and water, and reacting to obtain ONPF-biotin; the preparation method of AzONPF comprises the following steps: 1) Dissolving 2-azidoethanol in pyridine, adding chloroformyl- (4-nitro) -phenol ester, stirring uniformly, reacting, purifying to obtain compound 1, wherein the structural formula of the compound 1 is ; 2) Dissolving a compound 1 in dichloromethane, adding N-tertiary oxo-1, 2-ethylenediamine and triethylamine into a system, reacting at room temperature overnight, and after the reaction, further extracting and purifying the product to obtain a compound 2, wherein the compound 2 has the structural formula of ; 3) Dissolving the compound 2 in dichloromethane, adding trifluoroacetic acid, and reacting at room temperature to obtain a compound 3, wherein the compound 3 has the structural formula of ; 4) Dissolving 2-hydroxy-5-formyl-methyl benzoate in dimethylformamide, adding cesium carbonate, adding dimethylformamide solution of o-nitrobenzyl bromide under ice bath, heating to room temperature, reacting overnight, and further extracting and purifying to obtain compound 4 with structural formula of ; 5) Dissolving the compound 4 in methanol/tetrahydrofuran, slowly adding sodium borohydride under ice bath, stirring for continuous reaction, and further extracting and purifying the product after the reaction is finished to obtain a compound 5, wherein the compound 5 has the structural formula of ; 6) Dissolving compound 5 in tetrahydrofuran, adding aqueous solution of lithium hydroxide, reacting at room temperature overnight, further extracting and purifying the product after the reaction is finished, redissolving the obtained white crude product in dimethylformamide, adding 3, N-diisopropylethylamine, hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, reacting at room temperature overnight, further extracting and purifying the product after the reaction is finished to obtain white solid AzONPOH, wherein the structural formula of AzONPOH is ; 7) AzONPOH is dissolved in methylene dichloride, methylene dichloride solution of bis (2-methoxyethyl) amino sulfur trifluoride is slowly added in ice bath, the system is warmed to room temperature and reacted overnight, after the reaction is finished, the product is further extracted and purified to obtain white solid AzONPF, the structural formula of AzONPF is 。
  4. 4. The method according to claim 3, wherein the molar ratio of AzONPF to DBCO-biotin is 1:1, and the reaction condition is room temperature for 2 hours.
  5. 5. A space single cell transcriptome sequencing method based on light-controlled cell markers is characterized by comprising the following steps: 1) Labelling cells to be sequenced using ONPF-biotin as described in claim 2; 2) ONPF-biotin labeled cells were sorted and single cell sequenced.
  6. 6. The method according to claim 5, wherein the marking method in step 1) comprises: 11 Incubating the cells to be detected with a ONPF-biotin HBSS solution; 12 Removing the solution from the cells incubated in step 11), and performing laser irradiation; 13 After the cells were washed twice with the medium containing the streptavidin-fluorescent molecule conjugate, incubated at room temperature, and after the incubation, the cells were washed twice with HBSS solution again.
  7. 7. The method according to claim 6, wherein the number of labeling steps in step 1) is a plurality of times, and steps 11) to 13) are repeated after the last HBSS solution washing step 13).
  8. 8. The method according to claim 5, wherein said step 2) comprises the steps of: 21 Adding pancreatin into the marked cells, incubating, sucking out pancreatin, blowing off the cells with PBS containing 5% fetal calf serum, carrying out flow separation, observing obvious cell grouping in the flow signal, and selecting and separating positive communities; 22 Sorting positive cells into 96-well plates with pre-added lysate, one cell per well, and performing cell lysis; 23 Respectively carrying out reverse transcription on RNA obtained from each hole to obtain cDNA; 24 Respectively carrying out PCR amplification on the cDNA products obtained in the step 23); 25 Respectively purifying the PCR products of each hole by using magnetic beads, and constructing a second generation sequencing library; 26 Using 5200 Fragment Analyzer for quality control of the constructed library; 27 And carrying out secondary sequencing after merging the quality-controlled libraries, and carrying out subsequent analysis on about 140 tens of thousands of Reads samples in a total machine per sample to obtain a space single-cell transcriptome sequencing result.

Description

Space single cell transcriptome sequencing method based on light-controlled cell markers Technical Field The invention relates to the technical field of biological detection, in particular to a space single-cell transcriptome sequencing technology based on light-controlled cell markers. Background Single cell transcriptome sequencing is one of the core technologies of modern biology (scRNA-seq). By single cell sequencing, researchers can detect gene expression of each cell, thereby obtaining information on the type, number, state, etc. of the cells. The function and gene expression of each cell in a multicellular organism is regulated by its spatial location, but the spatial information of the cell is lost in the experimental procedure of single cell sequencing. In response to this challenge, researchers have developed a variety of methods that wish to achieve single cell sequencing with spatial resolution. The core of these methods is to perform fluorescent labeling of cells at specific spatial locations, then to isolate the labeled cells and to sequence single cells, thereby adding spatial location information to each cell in the single cell sequencing data. The Ido group of subjects utilizes genetically encoded light activated fluorescent proteins to achieve spatial labeling and single cell sequencing of cells in mouse tissue. The method relies on genetic manipulation and has higher application difficulty. The Costantino subject group adopts the photobleaching principle of fluorescein to realize the spatial labeling of cells, but the method has lower efficiency and high background signal. ZipSeq technology developed by Krummel task group utilizes light to control DNA hybridization on the cell surface, thereby realizing space-specific DNA bar code labeling and single cell sequencing. This method requires single-stranded DNA labelling of cells, and has a high background adsorption. The SCARI technology developed by Schumacher's task group uses a light activated genetic tag to circumvent single-stranded DNA markers, but cannot simultaneously mark multiple regions, limiting its range of application. The existing method has the problems of low labeling efficiency, small number of labeling positions, high background signal, dependence on genetic operation and the like, and limits the application of the method in the fields of biology, medicine and the like. Disclosure of Invention Spatial single cell transcriptome sequencing requires labeling cells at specific positions in space, whereas existing methods suffer from low labeling efficiency, a small number of labeling positions, high background signals, reliance on genetic manipulation, and the like. The method aims to solve the defects of the prior method by a brand-new chemical method and realize more efficient and practical space single-cell transcriptome sequencing. In order to solve the problems existing in the prior method, the invention is claimed to be applied to the space single cell transcriptome sequencing technology, and the structural formula of the ONPF-biotin is as follows: the invention provides a probe for sequencing a space single cell transcriptome, which is ONPF-biotin, wherein the structural formula of the ONPF-biotin is as follows: the invention provides a preparation method of a probe according to claim 2, which comprises the step of adding AzONPF and DBCO-biotin into a mixed solution of dimethyl sulfoxide and water for reaction to obtain ONPF-biotin. Wherein the mol ratio of AzONPF to DBCO-biotin is 1:1, and the reaction condition is that the reaction is carried out for 2 hours at room temperature. The preparation method of AzONPF comprises the following steps: 1) Dissolving 2-azidoethanol in pyridine, adding chloroformyl- (4-nitro) -phenol ester, stirring uniformly, reacting, purifying to obtain compound 1, wherein the structural formula of the compound 1 is 2) Dissolving the compound 1 in dichloromethane, adding N-tertiary oxo-1, 2-ethylenediamine and triethylamine into the system, reacting at room temperature overnight, and after the reaction, further extracting and purifying the product to obtain a compound 2; 3) Dissolving the compound 2 in dichloromethane, adding trifluoroacetic acid, and reacting at room temperature to obtain a compound 3; 4) Dissolving 2-hydroxy-5-formyl-methyl benzoate in dimethylformamide, adding cesium carbonate, adding dimethylformamide solution of o-nitrobenzyl bromide in ice bath, and heating the system to room temperature and reacting overnight; 5) Dissolving the compound 4 in methanol/tetrahydrofuran, slowly adding sodium borohydride under ice bath, stirring for continuous reaction, and after the reaction is finished, further extracting and purifying the product to obtain a compound 5; 6) Dissolving the compound 5 in tetrahydrofuran, adding an aqueous solution of lithium hydroxide, and reacting at room temperature overnight; after the reaction is finished, the product is further extracted and purified, th