Cell labeling methods

Abstract

The present invention pertains to a method for labelling a cell using a bar code. A method for labelling a cell using a bar code according to the present invention comprises (1) bringing, in a cell population including a plurality of single cells, a modified bar code directly or indirectly into contact with a cell surface protein of the cells, or (2) bringing a modified bar code directly or indirectly into contact with a cell surface protein of a single cell.

Description

[Technical field]

[0001] The present invention relates to a method for labeling cells with barcodes and a kit for use in said method.The present invention also relates to a method for multiplexed analysis of cell samples. [Background technology]

[0002] Comprehensive gene expression analysis is an essential technique for analyzing the characteristics of cells and tissues. By comparing gene expression profiles in various cells and tissues, it is possible to clarify the diversity of cell types present, the characteristics of various cells in different tissues, and the proportions in the population. In conventional bulk analysis, a large number of cells are grouped together, and mRNA is extracted from the mixed sample and subjected to expression analysis, so the average gene expression in those cells is observed. However, biological tissues are composed of a complex and diverse cell population. Therefore, in bulk analysis using the entire tissue, it is difficult to grasp the cellular state in the tissue and accurately interpret the expressed gene groups and their functions. In addition, even cultured cell lines that are thought to be homogeneous can be heterogeneous cell populations in the strict sense. Therefore, in bulk RNA-seq analysis, it is impossible to accurately grasp changes in gene expression that fluctuate due to, for example, the cell cycle or other modalities.

[0003] In order to overcome such problems, single cell RNA-seq analysis (scRNA-seq analysis) is a very useful technique (Non-Patent Document 1). scRNA-seq involves capturing a single cell (in this specification, unless otherwise specified, "single cell" is used to mean "a group of cells including a plurality of single cells or a single cell") and creating an RNA-seq library from each cell for transcriptome analysis. However, using a large number of single cells as samples requires a great deal of effort and huge cost, and until recently, scRNA-seq was not an easily applicable technique. However, droplet scRNA-seq using a microfluidic / microdroplet reaction system and scRNA-seq using microwells are currently being developed, and equipment for these techniques has been developed and is commercially available (for example, the BD Rhapsody system (Non-Patent Document 2), 10x Chromium (Non-Patent Document 3) 、 With such advances in single-cell analysis technology, it has become possible to prepare scRNA-seq libraries from more than 10,000 cells, significantly reducing the analysis cost per cell (Non-Patent Document 4).

[0004] However, although the cost per cell can be reduced, when multiple samples are analyzed or when time series analysis is performed at multiple time points, the overall analysis cost becomes high. Furthermore, when scRNA-seq experiments are performed on multiple samples at different times and compared, analysis errors (i.e., batch effect) may occur for each experimental batch (Non-Patent Document 5). Therefore, if multiple samples can be grouped together and processed simultaneously in one library preparation, not only can the analysis cost be significantly reduced, but the batch effect can be reduced and more accurate scRNA-seq analysis can be performed. The technology for grouping multiple samples together and analyzing them in one experimental operation is called "sample multiplexing."

[0005] In sample multiplexing, multiple cell populations are pre-labeled with barcode DNA tags with unique sequences corresponding to each cell, then mixed together for single-cell analysis. The resulting RNA-seq data can be used to trace the origin of each cell based on the barcode DNA sequence. In droplet scRNA-seq analysis or microwell scRNA-seq analysis, multiple cells may be captured in one compartment, and such multiple-cell contamination errors make data interpretation difficult. However, if different samples can be identified by different DNA tags, errors due to multiplets can be reduced. 。

[0006] Regarding multiplexing methods using this principle, for example, a method has been reported in which a cell labeling antibody that recognizes a specific cell surface protein is bound to a barcode oligo DNA (Non-Patent Document 6). However, the problem with this cell hashing method is that it is assumed that the cell surface protein is expressed in all cells in the target sample to be analyzed. Cell hash tag antibodies are antibody cocktails used for the purpose of comprehensively labeling cells originating from adult tissues (e.g., TotalSeq (registered trademark) anti-mouse hash tag antibody, BioLegend). In the case of hash tags, antibodies against CD45 and MHC class I are used (the former is for detecting cells of the immune system, and the latter is generally for detecting differentiated cells). However, for example, pluripotent stem cells, which are often used as a model of embryonic development, do not express CD45 or MHC class I, and the above method cannot be applied (Non-Patent Documents 7, 8). It has also been found that the above method cannot be applied to some non-embryonic cells such as chondrocytes.

[0007] Other labeling techniques for multiplexing have been developed, such as the transient barcode method (Non-Patent Document 11), which introduces barcode DNA into cells, and the cell tag index method (Non-Patent Document 12), which uses lentivirus to introduce barcode DNA. However, it is difficult to apply these methods to cells other than cultured cells, and therefore their versatility is limited. In addition, the ClickTag multiplexing method (Non-Patent Document 13), which combines click chemistry and NHS-ester chemistry, is a technique for attaching barcode DNA tags to cellular proteins, but the labeling efficiency of live cells is low, and this method can only be applied to methanol-fixed cells. The CellPlex method (3'CellPlex Kit, 10x Genomics) uses a reagent that introduces DNA tags into lipids in the cell membrane for universal cell labeling (Non-Patent Document 14), but it is difficult to apply it to methanol-fixed cells. Prior art documents Patent Literature

[0008] [Patent Document 1] WO 2020 / 010366 A1

[0009] [Non-patent document 1] X. Han, R. Wang, Y. Zhou, L. Fei, H. Sun, S. Lai, A. Saadatpour, Z. Zhou, H. Chen, F. Ye, D. Huang, Y. g, X. Jiang, J. Mao, Y. Chen, C. Lu, J. Xie, Q. Fang, Y. Wang, R. Yue, T. Li, H. Huang, SHOrkin, G.-C. Yuan, M. Chen, G. Guo, Mapping the mouse cell atlas by Microwell-seq,Cell 172(2018)1091-1107, https: / / doi.org / 10.1016 / j.cell.2018.02.001. [End Page 2]SJYoon,LSElahl,AMPasca,RMMarton,A.Gordon,O.Revah,Y.Miura,Y.Walczak,GMHoldgate,HCFan,JRHuguenard,DHGeschwind,SPPasca,Reliability of human cortical organoid generation.Nat.Methods 16(2019) 75-78, https: / / doi.org / 10.1038 / s41592-018-0255-0 [End Page 3]GXYZheng,JMTerry,P.Belgrader,P.Ryvkin,ZWBent,R.Wilson,SBZiraldo,TDWheeler,GPMcDermott,JZhu,MTGregory et.al.,Massively parallel digital transcriptional profiling of single cells,Nat.Commun.8(2017 Jan 16)14049; [Note 4]J.Cheng,J.Liao,X.Shao,X.Lu,X.Fan,Multiplexing methods for simultaneous large-scale transcriptomic profiling of samples at single-cell resolution,Adv.Sci.8(2021)2101229; [End Page 5]HTNTran,KSAng,M.Chevier,X.Zhang,NYSLee,M.Goh,J.Chen,A benchmark of batch-effect correction methods for single-cell RNA sequencing data,Genome Biol.21(2020)12, https: / / doi.org / 10.1186 / s13059-019-1850-9. [Non-Patent Document 6] M. Stoeckius, S. Zheng, B. Houck-Loomis, S. Hao, B. Z. Yeung, W. M. Mauck III, P. Smibert, R. Satija, Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics, Genome Biol. 19 (2018) 224, https: / / doi.org / 10.1186 / s13059-018-1603-1. [Non-Patent Document 7] S. L. McKinney-Freeman, O. Naveiras, F. Yates, S. Loewer, M. Philitas, M. Curran, P. J. Park, G. Q. Daley, Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells, Blood 114 (2009) 268 - 278, https: / / doi.org / 10.1182 / blood-2008-12-193888. [Non-Patent Document 8] A. S. Boyd, K. J. Wood, Variation in MHC expression between undifferentiated mouse ES cells and ES cell-derived insulin-producing cell clusters, Transplantation 87 (2009) 1300 - 1304, https: / / doi.org / 10.1097 / TP.ObO13e3181a19421. [End Page 9]T.Redmer,S.Diecke,T.Grigoryan,A.Quiroga-Negreira,W.Birchmeier,D.Besser,E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming,EMBO Rep. 12(2011) 720–726, https: / / doi.org / 10.1038 / embor.2011.88. [End Page 10]S.Shichino,S.Ueha,S.Hashimoto,T.Ogawa,H.Aoki,W.Bin,C.-Y.Chen,M.Kitabatake,N.Ouji-Sageshi ma,S.Hontsu,N.Sawabata,T.Kawaguchi,T.Okayama,E.Sugihara,T.Ito,K.Ikeo,T.Sato,K.Matsushima,TAS-Seq:a robust and sensitive amplification method for bead-based scRNA-seq,bioRxiv(2021),https: / / doi.org / 10.1101 / 2021.08.03.454735. [End Page 11]D.Shin,W.Lee,JHLee,D.Bang,Multiplexed single-cell RNA-seq via transient barcoding for simultaneous expression profiling of various drug perturbations,Sci.Adv.5(2019)eaav2249,https: / / doi.org / 10.1126 / sciadv.aav2249. [End Page 12]C.Guo,W.Kong,K.Kamimoto,GCRivera-Gonzalez,X.Yang,Y.Kirita,SAMorris,CellTag Indexing:genetic barcode-based sample multiplexing for single-cell genomics.Genome Biol.20(2019)90, https: / / doi.org / 10.1186 / s13059-019-1699. [End Page 13]J.Gehring,JHPark,S.Chen,M.Thomson,L.Pachter,Highly multiplexed single-cell RNA-seq by DNA oligonucleotide tagging of cellular proteins,Nat.Biotechnol.38(2020)35-38; [End Page 14]C.McGinnis,DMPatterson,J.Winkler,DNConrad,MYHein,V.Srivastava,JLHu,LMMurrow,JSWeissman,ZWerb,EDChow,ZJGartner,MULTI-seq:sample multiplexing for single-cell RNA sequencing using lipid-tagged indices,Nat.Methods 16(2019)619-626,https: / / doi.org / 10.1038 / s41592-019-0433-8. [Non-Patent Document 15] H. Niwa, S. Masui, I. Chambers, AG Smith, J. Miyazaki, Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3 / 4 in embryonic stem cells,Mol.Cell.Biol.22(2002)1526-1536,https: / / doi.org / 10.1128 / MCB.22.5.1526-1536.2002. [Non-patent document 16] K. Ogawa, H. Matsui, S. Ohtsuka, H. Niwa, A novel mechanism for regulating clonal propagation of mouse ES cells, Genes Cells 9(2004)471-477, https: / / doi.org / 10.1111 / j.1356-9597.2004.00736.x. [Non-patent document 17] M. Sugimoto, M. Kondo, M. Hirose, M. Suzuki, K. Mekada, T. Abe, H. Kiyonari, A. Ogura, N. Takagi, K. Artzt, K. Abe, Molecular identification of t▲w5▼:Vps52 promotes pluripotential cell differentiation through cell-cell interactions, Cell Rep.2(2012)1363-1374, https: / / doi.org / 10.1016 / j.celrep.2012.10.004. [Non-Patent Document 18] Package 'Seurat' January 14, 2022 Version 4.1.0, Title Tools for Single Cell Genomics, http: / / ftp1.us.debian.org / pub / cran / web / packages / Seurat / Seurat.pdf Summary of the Invention Problem to be solved by the invention

[0010] As a result of intensive research by the present inventors to solve the above problems, they developed a new cell labeling technique for multiplexed scRNA-seq analysis that does not depend on specific cell surface antigens, and came up with the present invention. One of the features of the method of the present invention is that it includes a step of directly or indirectly contacting a modified barcode with a cell surface protein of a cell. In one aspect, the present invention is a Universal Surface Biotinylation (USB) method. In another aspect, the method of the present invention is a method (single-step method) in which a barcode is directly bound to a cell surface protein of a cell. Means for solving the problem The present invention includes, but is not limited to, the following aspects.

[0011] [1] 1. A method for labeling cells with barcodes, comprising: (1) for a population of cells that includes a plurality of single cells, contacting, directly or indirectly, a cell surface protein of the cells with a modified barcode; or (2) contacting the cell surface proteins of a single cell directly or indirectly with the modified barcode; The method includes: [2] 1. A method for labeling cells with barcodes, comprising: (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins on single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; and, (ii) contacting each cell population or each single cell with a barcode-labeled biotin-binding substance; The method includes: [3] (ia) biotinylating a cell surface protein for a population of cells including a plurality of single cells; and, (ii) contacting each cell group with a barcode-labeled biotin-binding substance; The method according to claim 2, comprising: [4] The method according to [2] or [3], wherein in (i), an amino group, a sulfhydryl group, or a carboxyl group of a cell surface protein is biotinylated. [5] In (i), the reagent used for biotinylation is selected from the group consisting of sulfo-N-hydroxysuccinimide-biotin, N-hydroxysuccinimide-biotin, and pentafluorophenyl-biotin; wherein the bond between biotin and sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, or pentafluorophenyl may include a spacer; The method according to any one of [2] to [4]. [6] The method according to [5], wherein the reagent used for biotinylation in (i) has been frozen and stored. [7] The method according to any one of [2] to [6], wherein the biotin-binding substance is selected from the group consisting of streptavidin, avidin, and an anti-biotin antibody. [8] The method according to [7], wherein the biotin-binding substance is streptavidin. [9] The method according to any one of [1] to [8], wherein the barcode is an oligo-DNA.

[10] Labeling live cells, [1]-[9] Any one of the following The method according to

[11] A method according to any one of [1]-[9], for labeling fixed cells.

[12] a reagent for biotinylating cell surface proteins of a cell; and Barcoded biotin-binding substances A kit for use in a method for labeling cells with a barcode, comprising:

[13] The kit according to

[12] , wherein the reagent for biotinylating a cell surface protein of a cell is sulfo-N-hydroxysuccinimide-biotin.

[14] 1. A method for multiplexed analysis of a cell sample, comprising: (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins on single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; (ii) contacting each cell population or each single cell with a barcode-labeled biotin-binding substance; (iii) mixing all or a portion of the barcode-labeled cell population or single cells of (ii); and, (iv) Analyzing the mixture of cells (iii). The method includes:

[15] (ia) biotinylating a cell surface protein for a population of cells including a plurality of single cells; and, (ii) contacting each cell group with a barcode-labeled biotin-binding substance; Including, The method described in

[14] .

[16] The method according to

[15] , wherein the analysis is cellular RNA analysis.

[17] 1. A method for labeling cells with barcodes, comprising: (1) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (2) contacting a single cell with a barcode modified with a compound that binds to amino groups on a cell surface protein of the cell; The method includes:

[18] The method according to

[17] , wherein the compound that binds to an amino group of a cell surface protein of the cell is selected from the group consisting of sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, and pentafluorophenyl.

[19] The method according to

[18] , wherein the compound that binds to amino groups of cell surface proteins of cells has been frozen and stored.

[20] The method according to any one of

[17] to

[19] , wherein the barcode is an oligo-DNA. [twenty one] The methods described in

[17] -

[20] are used to label live cells. [twenty two] A method according to any one of

[17] -

[21] for labeling fixed cells. [twenty three] A kit for use in a method for labeling cells with a barcode, the kit comprising a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell. [twenty four] The kit according to

[23] , wherein the compound that binds to an amino group of a cell surface protein of a cell is sulfo-N-hydroxysuccinimide. [twenty five] 1. A method for multiplexed analysis of a cell sample, comprising: (1A) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (1B) contacting a single cell with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell; (2) Mixing all or a portion of the barcode-labeled cell population or single cells of (1); and, (3) Analyze the mixture of cells from (2). The method includes:

[26] The method according to

[25] , wherein the analysis is cellular RNA analysis.

[0012] The method of labeling cells with barcodes of the present invention, whether the USB method or the single-step method, targets cell surface proteins of cells, so that cells in any state can be labeled as long as the cell surface proteins are present. For example, since it does not depend on the expression of cadherin 1 (CDH1), cells in both undifferentiated and differentiated states can be labeled. of Any of these methods can be used as targets. In addition, there is no limitation on the type of cells to be labeled, and the method can be applied to any type of cell. Furthermore, the method can be applied to cells fixed with methanol or the like. Regarding the USB method, biotinylation does not depend on a specific cell surface protein and can be performed on any cell surface protein. The method of the present invention, which utilizes biotinylation of cell surface proteins, is a universal labeling technique that enables comprehensive labeling of cells. The technique of the present invention (hereinafter referred to as the "USB method") is highly versatile and can be applied to any type of cell.

[0013] The method of the present invention allows the labeling of all types of cells by utilizing biotin, which can be introduced into any cell surface protein. The USB method has no adverse effects on the growth or colony-forming ability of the tested cells. Furthermore, when sample multiplexing is performed and the USB method is applied to single-cell analysis, it has been shown in an experiment modeling the differentiation process of mouse embryonic stem (ES) cells that various differentiated cells can be successfully captured without adversely affecting the gene detection power or transcriptome acquisition in each cell. The USB method can be used for many types of cells, including human iPS-derived chondrocytes and rat lung-derived cells, which have not been successfully applied to conventional cell hashing methods until now. The USB method can theoretically be applied to any type of cell, and is a simple, highly efficient, and cost-effective method for multiplexing scRNA-seq.

[0014] The single-step method includes a step of contacting a barcode modified with a compound that binds to the amino group of a cell surface protein of a cell. As with the USB method, the single-step method also targets cell surface proteins of cells, allowing labeling of all types of cells. The single-step method has no adverse effects on the growth or colony-forming ability of the tested cells. Furthermore, it has been confirmed in the examples herein that when sample multiplexing is performed and the single-step method is applied to single-cell analysis, various differentiated cells can be successfully captured without adversely affecting the detection power of genes in each cell or the acquisition of the transcriptome. 。

[0015] The present invention can be used for disease analysis, such as performing scRNA-seq on cancer tissues. The present invention can also be used to confirm whether iPS cells have differentiated into a target type of cell in transplantation medicine using iPS cells. The present invention can also be used to verify the effect of a drug on patient-derived iPS cells when screening candidates for therapeutic drugs for genetic diseases. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 illustrates the principles of USB and antibody methods (Ab methods). In the USB method, cell surface proteins (blue) are universally biotinylated (reddish brown) with S-NHS-biotin and then treated with barcode DNA-labeled streptavidin. In the Ab method, cells are treated with biotin-conjugated antibodies against specific cell surface proteins (red), followed by treatment with barcode DNA-labeled streptavidin. If a particular protein is not expressed in some cells, those cells will not be labeled by the Ab method, but all cells can be labeled by the USB method. [Figure 2] Figure 2 shows the results of flow cytometry of undifferentiated ES cells and differentiated cells labeled by the USB and Ab methods. The vertical axis of Figure 2 shows the number of cells, and the horizontal axis shows the fluorescence intensity of PE (phycoerythrin). Flow cytometry showed that the USB method had a very high efficiency of cell labeling, with 99.6% of undifferentiated cells (upper left) and 96.4% of differentiated cells (upper right) being PE positive. In contrast, when the Ab method was used, 87.8% of undifferentiated cells (lower left) and 9.9% of differentiated cells (lower right) were PE positive. [Figure 3] Figure 3 shows fluorescent micrographs (magnification x200) of cells labeled by the USB method (upper panel) and the Ab method (lower panel). An antibody against mouse CDH1 was used for the Ab method. CDH1 is known to be expressed in undifferentiated ES cells, but expression was limited to cells in an undifferentiated state. (Top) USB method, (bottom) Ab method. (Left) Undifferentiated ES cells, (right) Differentiated cells (12 days after differentiation induction). Ph A, phase contrast image; PE, fluorescence image of TotalSeq®-PE-streptavidin; left arrow head, PE-negative cell; right arrow, PE-positive cell. [Figure 4] Figure 4 shows the results of scRNA-seq analysis of ES cells and their differentiated derivatives. For cell labeling, USB multiplexing or Ab multiplexing using CDH1 antibody was used. (A) UMAP (uniform manifold approximation and projection) analysis classified the cells into 11 clusters: 0: pluripotent cells; 1: naive cells; 2: vascular smooth muscle cells; 3: Prime Type (B) Distribution of cell samples on the UMAP. The color code of each sample is shown below the figure. (C) Distribution of Cdh1-positive cells on the UMAP. [Figure 5] Figure 5 shows a comparison of the numbers of cells detected by the USB method and the Ab method in the Cdh1-positive (left) and Cdh1-negative (right) clusters in Figure 4. [Figure 6] Figure 6 shows the results of CDH1 immunostaining for (A) undifferentiated ES cells and (B) differentiated cells. CDH1 signals were observed on the cell surface of all undifferentiated ES cells, but were detected only in a small number of differentiated cells. Green, CDH1; red, OCT4; blue, DAPI (nuclei). [Figure 7] Figure 7 shows that S-NHS-biotin treatment does not affect the viability of undifferentiated ES cells. Checked The results are shown below. (Right) Cells treated with S-NHS-biotin and (left) untreated control cells. (Bottom) Enlarged image x7.3. [Figure 8] Figure 8 shows the quality check by electrophoresis of the whole transcriptome cDNA (A) amplified by the TAS-seq protocol and the barcode DNA (B) amplified by the TAS-seq protocol. The electropherograms showed that the yield and size distribution of the cDNA and barcode DNA were sufficient for further analysis. [Figure 9] Figure 9 shows the results of examining the distribution of undifferentiated ES cells and differentiated cells labeled by the USB and Ab methods using the UMAP method: (A) UMAP distribution of undifferentiated ES cells labeled by the USB method; (B) UMAP distribution of undifferentiated ES cells labeled by the Ab method; (C) UMAP distribution of differentiated cells labeled by the USB method; and (D) UMAP distribution of differentiated cells labeled by the Ab method. [Figure 10] Figure 10 shows the results of scRNA-seq analysis of multiplexed samples. A total of six samples were used: four samples of undifferentiated and differentiated cells derived from R1 ES cells labeled by either the USB or Ab method, and two samples of undifferentiated and differentiated cells derived from EB3 ES cell line labeled by the USB method. Cell clusters were analyzed by the UMAP method, and the distribution of each sample is shown in (A). (B) Eleven clusters were identified as follows: 0: vascular smooth muscle; 1: pluripotent cells; 2: naive ES cells; 3: epithelial cells; 4: dopamine neurons; 5: Prime Type 5: Pluripotent stem cells; 6: Macrophages; 7: MEF (feeder cells); 8: Neurons; 9: Glial cells; 10: Vascular endothelial cells. (C) Cdh1 expression levels are overlaid onto the UMAP distribution in (A). [Figure 11] Figure 11 shows the results of flow cytometry analysis after labeling, using the USB method, chondrocytes that cannot be labeled using the Ab method that uses existing reagents. The vertical axis of Figure 11 shows side scatter signal (SSC), and the horizontal axis shows the fluorescence intensity of PE (phycoerythrin). Chondrocytes derived from mouse growth plates were labeled with MHC class I antibody (left), CD45 antibody (center), and the USB method (right), and analyzed by flow cytometry. Mouse growth plate chondrocytes that are hardly stained with MHC and CD45 antibodies are efficiently labeled by the USB method. [Figure 12] Figure 12, like Figure 11, shows the results of flow cytometry analysis after labeling rat cells that cannot be labeled by the Ab method using existing reagents using the USB method. The upper part of Figure 12 shows the results of flow cytometry analysis of rat lung cells double-stained with PE-labeled CD31 antibody and PerCP-labeled CD45 antibody, and the lower part of Figure 12 shows the results of flow cytometry analysis verifying the cell labeling efficiency by the USB method in CD31-positive cells, CD45-positive cells, and double-negative cells. In the experiment to verify the cell labeling efficiency by the USB method, APC-labeled streptavidin was used. The vertical axis of the upper part of Figure 12 shows the fluorescence intensity of PE, the horizontal axis shows the fluorescence intensity of PerCP, and the vertical axis of the lower part of Figure 12 shows the side scatter signal (SSC), and the horizontal axis shows the fluorescence intensity of APC. Lung cells dissociated from rat lungs were fractionated into three populations: CD31-positive, CD45-positive, and double-negative. It was found that all the fractionated cells were labeled by the USB method. Rats are a species for which there are no commercially available sample multiplexing kits based on Ab methods. [Figure 13] Figure 13 shows the results of verifying the presence or absence of non-specific binding of antibodies by flow cytometry analysis. The vertical axis of Figure 13 shows the number of cells, and the horizontal axis shows the fluorescence intensity of PE (phycoerythrin). (A) Unlabeled undifferentiated ES cells; (B) Undifferentiated ES cells treated with biotin-isotype control antibody and TotalSeq (registered trademark)-streptavidin (PE-bound). No non-specific binding of the isotype control antibody was detected by flow cytometry analysis. [Figure 14] Figure 14 shows non-specific binding of isotype control antibodies and barcode-tagged streptavidin (TotalSeq (registered trademark)-streptavidin) detected by the TAS-seq method. A cDNA synthesis reaction was carried out for each sample, and barcode DNA was amplified by the TAS-seq method. The samples were then analyzed using an Agilent Bioanalyzer. (A) Antibodies and TotalSeq (Registered Trademark) (B) cells not treated with streptavidin (negative control sample); (C) cells treated with isotype control antibody followed by TotalSeq®-streptavidin, and (D) cells treated with biotin-modified anti-CDH1 antibody followed by TotalSeq®-streptavidin (positive control sample). The relationship between the sample reagents is shown at the bottom of each panel (A)-(D) (see also Figure 1). No amplification of barcode DNA was observed in the negative control sample (A), but a clear peak (arrow) was observed in the positive control sample (D). A peak was observed in the other samples (B and C), although it was smaller than that in (D) (arrow), confirming the presence of nonspecific binding of TotalSeq®-streptavidin and / or isotype control antibody. Using the highly powerful TAS-seq method, we confirmed low levels of nonspecific binding of TotalSeq®-streptavidin and / or antibodies that could not be detected by flow cytometry analysis. Although nonspecific binding of TotalSeq®-streptavidin and / or antibodies does not pose an obstacle to sample multiplexing in scRNA-seq analysis, this indicates that the USB method is superior to the Ab method in terms of technical rigor. [Figure 15] Figure 15 shows the results of labeling cells using sulfo-NHS-esterified oligo-DNA (NEO) (single-step method) and verifying the labeling efficiency by flow cytometry analysis. 6-FAM (6-carboxyfluorescein) was added to the 3' end of NEO used to verify the labeling efficiency in the single-step method. The vertical axis of Figure 15 shows the number of cells, and the horizontal axis shows the fluorescence intensity of 6-FAM (6-carboxyfluorescein). [Figure 16] Figure 16 shows the results of multiplex scRNA-seq analysis of various types of cells labeled by the single-step method. [Figure 17] Figure 17 is a bar graph showing the number of each of the nine types of cells detected by the multiplex scRNA-seq analysis in Figure 16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention includes the following embodiments, but is not limited thereto. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. The substances, materials and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in the present specification, it means that the embodiment is not limited, i.e., it is non-limiting.

[0018] In one aspect, the present invention relates to a method for labeling cells with a barcode. (1) for a population of cells that includes a plurality of single cells, contacting, directly or indirectly, a cell surface protein of the cells with a modified barcode; or (2) contacting the cell surface proteins of a single cell directly or indirectly with the modified barcode; This includes:

[0019] One feature of the method of the present invention is that a cell surface protein of a cell is directly or indirectly contacted with a modified barcode.

[0020] In one aspect, the present invention is a Universal Surface Biotinylation (USB) method. In the USB method, cell surface proteins of cells are indirectly contacted with a barcode (through biotin-biotin-binding substance). In the USB method, cell surface proteins are biotinylated, and the barcode is modified with a biotin-binding substance. In the USB method, a "modified barcode" means a barcode to which a biotin-binding substance has been added, i.e., a barcode-added biotin-binding substance.

[0021] In another aspect, the method of the invention teeth, This is a method of directly binding a barcode to a cell surface protein of a cell (single-step method (or direct method)). In the single-step method, the cell surface protein of the cell is directly contacted with the modified barcode. In the single-step method, the barcode is modified with a compound that binds to the amino group of the cell surface protein of the cell. In the single-step method, "modified barcode" means a barcode modified with a compound that binds to the amino group of the cell surface protein of the cell.

[0022] 1. Method for labeling cells with barcodes (1) (USB method) In one aspect, the present invention relates to a method for labeling cells with a barcode, the method including, but not limited to, (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins on single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; and, (ii) contacting each cell population or each single cell with a barcode-labeled biotin-binding substance; This includes: The method includes biotinylating a cell surface protein present on the cell surface. Biotinylation can be applied to any cell surface protein, and can comprehensively label cells regardless of the type of cell. In other words, since the labeling does not depend on a specific cell surface protein, the type of cells that can be the subject of the method is not limited. Animal cells, plant cells, cultured cells, and all other cells can be the subject. The method first involves biotinylating cell surface proteins of a cell. (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins on single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells. Biotinylation of proteins may be performed on a cell group including a plurality of single cells or on a single cell, or cell surface proteins of a plurality of cells may be biotinylated and then the biotinylated cells may be separated into a cell group including a plurality of single cells or into single cells. (ii) Prior to the step of contacting with a barcode-added biotin-binding substance, it is necessary that the cells are separated into a cell group including a plurality of single cells or into single cells. Preferably, biotinylation of proteins is performed on a cell group including a plurality of single cells or on a single cell. A "cell group containing a plurality of single cells" is a cell group that is a mass of single cells that share common characteristics or origin, such as a cell group consisting of a plurality of single cells with common characteristics or a cell group consisting of a plurality of single cells obtained from a common organ. In one embodiment, the method comprises: (ia) biotinylating a cell surface protein for a population of cells including a plurality of single cells; and, (ii) contacting each cell group with a barcode-labeled biotin-binding substance; This includes: A method for isolating a cell group containing a plurality of single cells or a single cell from a cell mass (e.g., an organ, etc.) containing a plurality of types of cells can be a known method and is not particularly limited. Those skilled in the art can use known methods depending on the cell mass, the type of cell contained in the cell mass, the cell group containing a plurality of single cells to be obtained (labeled) and isolated, or the type of single cell. The same applies when isolation is performed after biotinylation. Biotin is a water-soluble vitamin classified as vitamin B. 7 Biotinylation is the process of attaching biotin to proteins and macromolecules. Biotinylation is Biotin Labeling Reagents There is a method of chemically binding a functional group on a protein, such as an amino group, a sulfhydryl group, a carboxyl group, or an aldehyde group (oxidation of sugar chains), using a biotin-labeling reagent. Many types of biotin-labeling reagents are known, and those skilled in the art can select an appropriate biotin-labeling reagent based on the functional group to be used, the hydrophilicity of the labeling reagent, whether or not it is cleaved after the labeling reaction, and other factors. Biotin Labeling Reagents can be selected. Amino groups (mainly primary amines: -NH 2 ) is a lysine residue found in many proteins. Side Chain and amino termini. The ε-amino group of lysine residues is particularly highly reactive, and lysine residues are often present on the surface of the tertiary structure of proteins, making the amino group a particularly useful functional group for biotinylation. A representative amino group-reactive biotin labeling reagent is biotin with an N-hydroxysuccinimide (NHS) ester. NHS esters react with amino groups to form amide bonds. NHS ester biotin includes sulfo-NHS ester biotin, which has a sulfonic acid group, and NHS ester biotin, which does not have a sulfonic acid group. The former includes, for example, sulfo-N-hydroxysuccinimide-biotin, and the latter includes, for example, N-hydroxysuccinimide-biotin. Also, a long spacer between biotin and NHS-ester can be used. The length of the spacer is not limited to 0 to 300 Å, preferably 10 to 50 Å. Alternatively, a spacer having PEG (polyethylene glycol) introduced into the spacer portion (e.g., NHS-PEG4-biotin) can be used. Alternatively, a type that can appropriately cleave biotin can be used. The bond between biotin and the labeled protein can be cleaved by the spacer in some cases. Other than these, pentafluorophenyl-biotin and biotinylated isothiocyanate can be used for biotinylation of amino groups. Pentafluorophenyl-biotin and biotinylated isothiocyanate can bind to both primary and secondary amines. Sulfhydryl groups (-SH) of cysteine ​​residues can also be used for biotinylation. In particular, when an amino group is present at the active site of a protein and there is concern that labeling of the amino group may inactivate the protein, sulfhydryl groups may be biotinylated. However, when performing the labeling reaction, the sulfhydryl groups must be in a reduced state, that is, they must not have formed disulfide bonds (SS bonds) through oxidation. If there are no reduced sulfhydryl groups, the disulfide bonds are cleaved using a reducing agent. If the cell surface protein does not contain a cysteine ​​residue, a sulfhydryl group may be generated by modifying the lysine residue and used. Examples of sulfhydryl-reactive biotin labeling reagents that can be used include biotin having a maleimide group and biotin having a bromoacetamide group. The carboxyl group (-COOH) of aspartic acid and glutamic acid Side Chain In addition, it is present at the carboxyl terminus. When targeting the carboxyl group, an amino group (-NH 2 ) or hydrazide-derivatized biotin to form an amide bond. Propyl ]carbodiimide hydrochloride) and the like can be used. The cis-diol of sialic acid on the glycan can be synthesized by, for example, sodium periodate (NaIO 4 ) to generate an aldehyde group (-CHO), which can react with hydrazides to form hydrazone bonds. Glycoproteins can be labeled with hydrazide-derivatized biotin by mild oxidation to generate aldehyde groups. Alternatively, non-specific biotinylation can be achieved by using photoreactive biotin compounds that react non-specifically upon exposure to ultraviolet (UV) light. For example, photoreactive biotin labeling reagents that contain an aryl azide group are activated by UV light irradiation to produce aryl nitrenes (half-lives of 10 -4 Photoreactive biotin compounds react nonspecifically with nearby double bonds, hydrogen bond regions with high electron density, amino groups, and sulfhydryl groups. The use of photoreactive biotin compounds is useful when nonspecific labeling is required. In one embodiment, in (i), an amino group, a sulfhydryl group, or a carboxyl group of a cell surface protein is biotinylated. In one embodiment, the amino group of the cell surface protein is biotinylated. In one embodiment, in (i), the reagent used for biotinylation (biotin labeling reagent, reagent for biotinylating the cell surface protein of the cell) is selected from the group consisting of sulfo-N-hydroxysuccinimide-biotin, N-hydroxysuccinimide-biotin, and pentafluorophenyl-biotin. The bond between biotin and sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, or pentafluorophenyl may contain a spacer. The length of the spacer is, but is not limited to, 0 to 300 Å, preferably 10 to 50 Å. In one embodiment, In (i), the reagent used for biotinylation is selected from the group consisting of sulfo-N-hydroxysuccinimide-biotin, N-hydroxysuccinimide-biotin, and pentafluorophenyl-biotin; Here, the bond between biotin and sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, or pentafluorophenyl may contain a spacer. In one embodiment, the concentration of the reagent used for biotinylation can be appropriately applied depending on the type of cells used. The concentration of S-NHS-biotin in the example was 10 μg / mL, but it can be, for example, 1-50 μg / mL depending on the type of cells used. In one embodiment of the method, the reagent used for biotinylation in (i) may be cryopreserved. For example, sulfo-N-hydroxysuccinimide-biotin may be prepared in advance and cryopreserved for use whenever necessary. The method includes (i) biotinylating cell surface proteins, and then (ii) contacting each cell group or each single cell with a barcode-labeled biotin-binding substance. The "biotin-binding substance" is not particularly limited as long as it has the property of binding to biotin. It is preferable that the substance has little or no effect on cell growth and survival when bound to a cell surface protein via biotin. Non-limiting examples of biotin-binding substances include streptavidin, avidin, anti-biotin antibodies, etc. In one embodiment, the biotin-binding substance is selected from the group consisting of streptavidin, avidin, anti-biotin antibodies, etc. In one embodiment, the biotin-binding substance is streptavidin. A "barcode" is a substance that contains information for identifying each cell group or each single cell. The barcode can be added to a biotin-binding substance, and the type is not particularly limited as long as the barcode is a substance that contains information that can identify each cell group or each single cell. Non-limiting examples of barcodes include oligo-DNA and oligo-RNA. In one embodiment, the barcode is oligo-DNA. A "barcode-attached biotin-binding substance" is a substance in which a barcode (e.g., oligo-DNA) is attached to a biotin-binding substance (e.g., streptavidin). A detection label may be further attached to the barcode-attached biotin-binding substance. For example, by attaching a fluorescent substance to the barcode-attached biotin-binding substance, it becomes possible to analyze the labeled cells by flow cytometry. Alternatively, by attaching an epitope containing a specific protein or the like to the barcode-attached biotin-binding substance and detecting it by fluorescence or the like using an antibody or the like that binds to the epitope, it becomes possible to perform immunofluorescence analysis. The method of contacting each cell group or each single cell with a barcode-attached biotin-binding substance is not particularly limited. For example, the method is carried out by suspending each cell group or each single cell to which biotin has been attached in a solution containing a barcode-attached biotin-binding substance. In one embodiment of the method, the cells to be labeled may be live cells in vitro or in vivo. Alternatively, the cells to be labeled may be cells that have been fixed before biotinylation. In one embodiment of the method, the cells to be labeled may be cells that have been fixed and stored before biotinylation. The fixation and storage of the cells may be performed by a known method depending on the type of cells. For example, cells that have been stored at a low temperature (e.g., −80° C.) after fixing with methanol may be used. Alternatively, the cells may be cells that have been cryopreserved before biotinylation. The cryopreservation of the cells may be performed by a known method depending on the type of cells. The method may be in vivo, ex vivo or in vitro, preferably an in vitro method.

[0023] 2. Kit (1) The present invention also relates in one aspect to a kit for use in the method for labeling cells with barcodes. A reagent for biotinylating cell surface proteins, and Barcoded biotin-binding substances Includes. In one embodiment, the kit can be used in "1. Method for labeling cells with barcodes (1)" or "3. Method for multiplexed analysis of cell samples (1)". The "reagent for biotinylating cell surface proteins of cells" (reagents used for biotinylation, biotin-labeled reagents), "barcodes," "biotin-binding substances," and "barcode-added biotin-binding substances" are as explained in "1. Method for labeling cells with barcodes (1)." In one aspect 、 A reagent for biotinylating cell surface proteins is sulfo-N-hydroxysuccinimide-biotin. In one embodiment, the "barcode" is an oligo-DNA. In one embodiment, the "biotin-binding substance" is streptavidin.

[0024] 3. Multiplexed analysis method for cell samples (1) The present invention further relates to a method for multiplexed analysis of a cell sample, including, but not limited to, the steps of: (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins on single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; (ii) contacting each cell population or each single cell with a barcode-labeled biotin-binding substance; (iii) mixing all or a portion of the barcode-labeled cell population or single cells of (ii); and, (iv) Analyzing the mixture of cells (iii). This includes: (ia), (ib), (ic), and (ii) are as explained in “1. Method for labeling cells with barcodes (1)”. The method for multiplexing analysis of a cell sample further comprises, after step (ii), (iii) mixing all or a portion of the barcode-labeled cell population or single cells of (ii); and, (iv) Analyzing the mixture of cells (iii). This includes: In the above method, each cell group or each single cell is labeled with an identifiable barcode, and therefore, after labeling, all or part of the barcode-labeled cell groups or single cells can be mixed and analyzed together. That is, even if analyzed together, it is possible to identify which cell group or single cell the result is for by the barcode that can identify each cell group or single cell. The analysis is not limited as long as it is a means for analyzing cells. The analysis subject includes the contents of the cell (e.g., nucleic acid such as DNA and RNA). In one embodiment, the analysis is RNA analysis of the cell. By directly or indirectly (secondarily) labeling the barcode-added biotin-binding substance with fluorescence or the like, flow cytometry, immunofluorescence analysis, and the like can be performed. Alternatively, cells with similar properties can be classified into multiple clusters using the UMAP (uniform manifold approximation and projection) method based on the data obtained by scRNA-seq analysis. UMAP is an analysis tool that performs dimensionality reduction (dimensional compression) on a given sequence of points, and visualizes points that are "close" in high dimensions by arranging them "close" in low dimensions. By identifying the sample from which it originates using a barcode that is different for each sample as an index, it is also possible to clarify the differences in the characteristics of each sample by comparing the differences between samples in the number of cells contained in each classified cluster. It is also possible to sequence a mixture of all or part of a barcode-labeled cell group or single cell collectively, and then use the barcodes to distinguish which cell group or single cell the sequence belongs to and analyze them individually. In addition to RNA analysis, it is also expected to be applied to genome DNA analysis. For example, by performing genome DNA sequencing on a single cell and detecting DNA base sequence mutations for each single cell, it is possible to create a cell division lineage of each cell. When performing genome DNA sequencing on a single cell, sample multiplexing by adding a barcode can be performed, which can significantly reduce the analysis costs involved in creating a cell division lineage of a single cell group derived from multiple tissues. In addition, for example, quantitative detection of mitochondrial DNA by DNA sequencing on a single cell can be performed, which can track the fluctuation in the number of mitochondria within a cell group. In this case, sample multiplexing by adding a barcode can reduce the analysis costs. In one embodiment, the method for multiplexing a cell sample comprises: (ia) biotinylating a cell surface protein for a population of cells including a plurality of single cells; and, (ii) contacting each cell group with a barcode-labeled biotin-binding substance; This includes: We have developed a new, simple and reliable cell labeling method for multiplexing in scRNA-seq analysis. Conventional cell hashing methods target specific proteins that are widely expressed on the surface of the cells being tested, and are therefore difficult to use when such proteins are not widely expressed in the sample. However, the USB method does not require such specific proteins and uses a universal label that can target any protein, overcoming the shortcomings of existing techniques. Currently available multiplex reagents use antibodies against cell surface proteins such as CD298 and / or MHC class I antigens or CD45, as these antigens are believed to be universally expressed in adult tissues or the immune system. However, embryonic cells do not express these proteins, so the current cell hashing method is not applicable. In contrast, the USB method is compatible with all cell surface proteins and can be used for multiplexing regardless of cell type (as long as the cell has the surface protein). In fact, we have successfully applied the USB method to multiplex chondrocytes derived from mouse, monkey, and human iPS cells, and rat lung cells, which could not be studied by Ab methods using CD45 or MHC antibodies (Figures 11 and 12). Conventional labeling techniques for multiplexing, such as the transient barcode method, which introduces barcode DNA into cells, and the cell tag index method, which uses lentivirus to introduce barcode DNA, are difficult to apply to cells other than cultured cells, and therefore their versatility is limited. In contrast, the USB method is highly versatile and can be applied to any type of cell. The USB method is applicable to animals other than mice and humans. In other species, information on cell surface proteins or antibodies against them may not be readily available, and therefore it is difficult to apply traditional Ab methods. However, the USB method has been proven to be applicable to monkeys and rats, and theoretically it can be applied to cells of any species. In addition, the ClickTag multiplexing method (Non-Patent Document 13), which combines click chemistry and NHS-ester, is a technique for attaching barcode DNA tags to cellular proteins, similar to the USB method. However, the labeling efficiency of live cells is low, and this method can only be applied to methanol-fixed cells. The CellPlex method (3'CellPlex Kit, 10xGenomics) uses a reagent that introduces DNA tags into lipids in the cell membrane for universal cell labeling (Non-Patent Document 14), but it is difficult to apply it to methanol-fixed cells. In contrast, the USB method can also be applied to fixed cells, allowing for more flexible sample multiplexing. As described in the Materials and Methods section of the Examples, all components used in the USB method are commercially available and relatively inexpensive. The stock solution of S-NHS-biotin is stable for more than one year at -80°C, and a vial containing 50 mg of S-NHS-biotin is Up to 10,000 samples 10 micrograms of DNA barcoded streptavidin can be used for more than 150 multiplex experiments. 10 types of DNA barcoded streptavidin are commercially available, so 10-plex analysis can be performed at once. Streptavidin with any barcode oligo DNA can be custom-made, so it is possible to perform more than 10-plex multiplex experiments. In summary, the USB method is a cost-effective method that makes single-cell multiplex analysis more affordable, and the USB method is a universal labeling method that can be applied to either live or fixed cells, greatly facilitating all kinds of multiplexed scRNA-Seq analysis. Compared to the single-step method, the USB method has advantages such as being easy to introduce since existing reagents can be used, and the cost per sample is lower than that of the single-step method. The method may be in vivo, ex vivo or in vitro, preferably an in vitro method.

[0025] 4. Direct labeling of cells with barcodes (2) (single-step method) In one aspect, the present invention relates to a method for labeling cells with a barcode, the method including, but not limited to, (1A) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (1B) contacting a single cell with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell; This includes: The terms "cell group including a plurality of single cells", "cell", "barcode", "cell surface protein", etc. are as explained in "1. Method for labeling cells with barcodes (1)". By contacting a barcode modified with a compound that binds to the amino group of a cell surface protein of a cell with the cell, the compound in the modified barcode that binds to the amino group of the surface protein binds to the amino group of the cell surface protein, and the cell is labeled with a barcode. In one embodiment, the compound that binds to amino groups of cell surface proteins of cells is selected from the group consisting of sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, and pentafluorophenyl. Amino groups (mainly primary amines: -NH 2 ) is a lysine residue found in many proteins. Side Chain and amino termini. In particular, the ε-amino group of lysine residues is highly reactive, and lysine residues are often present on the surface of the tertiary structure of proteins, making the amino group a particularly accessible functional group for protein modification. A representative compound that binds to the amino group of cell surface proteins of cells is N-hydroxysuccinimide (NHS)-ester. NHS-ester barcodes react with the amino group of cell surface proteins to form amide bonds. NHS-esters include sulfo-NHS-esters that have a sulfonic acid group and NHS-esters that do not have a sulfonic acid group. The former includes, for example, sulfo-N-hydroxysuccinimide (sulfo-NHS), and the latter includes, for example, N-hydroxysuccinimide (NHS). It is also possible to provide a spacer between the barcode and the NHS-ester. The length of the spacer is not limited to 0 to 300 Å, and preferably 10 to 50 Å. Alternatively, a spacer having PEG (polyethylene glycol) introduced into the spacer portion (e.g., NHS-PEG4) can also be used. The method for binding the barcode to the NHS-ester is not particularly limited, and any method can be used. In one embodiment, 1-ethyl-3-[3-dimethylamino Propyl ]Carbodiimide hydrochloride (EDC), N,N' A carbodiimide compound such as -dicyclohexylcarbodiimide is used. By incorporating sulfo-NHS or NHS into the EDC conjugation protocol, the reaction efficiency is increased and an intermediate with dry stability (amine reactivity) is generated. Specifically, for example, an oligo-DNA (barcode) modified with a carboxyl group (-COOH) at the 5' end is first prepared. The addition of a carboxyl group to the oligo-DNA can be performed by a known method. For example, but not limited to, 10-carboxy-decyl-(2-cyanoethyl)(N,N-diisopropyl)-phosphoramidite, N-hydroxysuccinimide ester (e.g., reagent 5'-Carboxy-Modifier C10 (Glen Research)) may be used. Then, NHS is attached to the -COOH of 5'-COOH-DNA by EDC to form an NHS-ester, which is much more stable than the O-acylisourea intermediate. The "(sulfo-)NHS-esterified" DNA (activated state) forms a stable bond with an amino group in a cell surface protein. do. This results in labeling of cells with (sulfo-)NHS-esterified DNA (modified barcode). Alternatively, "(sulfo-)NHS-esterified" DNA may be prepared by the following method. For example, -NH 2 The modified oligo DNA was treated with disuccinimidyl suberate (DSS) (Thermo Fisher Scientific (trademark), 21655, etc.) or bis(succinimidyl) suberate (BS3). (Thermo Fisher Scientific (trademark), 21580, etc.) to induce "(sulfo)NHS-esterified" DNA. Specifically, for example, the 5' end is first converted to an amino group (-NH 2 Prepare an oligo DNA (barcode) modified with -NH 2The addition can be carried out by known methods, such as, but not limited to, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (e.g., the reagent 5'-Amino-Modifier C6 (Glen Research) Then, the 5' end may be modified with -NH 2 The DNA to which the -NHS-ester has been added is reacted with DSS or BS3. DSS is a reagent that has two NHS-esters. By reacting with DNA, one of the NHS-esters becomes -NH 2 BS3 is a DSS with a sulfo group added, and when it reacts with DNA in the same way as DSS, one of the NHS esters becomes -NH 2 The other end is exposed, and an activated NHS-ester is added to the DNA end. In this method, once two DNAs bind to one DSS or BS3, they are no longer able to bind to cells. Therefore, it is necessary to adjust the reaction conditions (reaction time, etc.) so that only one DNA binds to DSS or BS3. Other examples of barcodes that can be used to modify barcodes include pentafluorophenyl isothiocyanate, which can be used to bind to amino groups on cell surface proteins. Pentafluorophenyl isothiocyanate can bind to both primary and secondary amines. In addition, 6-FAM (6-carboxyfluorescein), biotin, etc. may be bound to the non-NHS-ester modified portion of the barcode to be used for detection other than multiplex analysis, etc. For example, when the 5' end of an oligo DNA is modified with NHS-ester, 6-FAM or biotin may be added to the 3' end of the DNA. In one embodiment, the compound that binds to amino groups on cell surface proteins of cells has been cryopreserved. In one embodiment, the barcode is an oligo-DNA. In one embodiment, the method comprises labeling live cells. 。In one embodiment, the method labels fixed cells. The terms "live cells" and "fixed cells" are as described in "1. Method for labeling cells with barcodes (1)". The method may be in vivo, ex vivo or in vitro, preferably an in vitro method. The method may be in vivo, ex vivo or in vitro, preferably an in vitro method. In addition, unless there are any technical problems, the contents explained in "1. Labeling cells with barcodes (1) (USB method)" are applicable to this section. 「 4. Direct labeling of cells with barcodes (2) (single-step method) 」 This also applies to. The single-step method simplifies the work process because the cell surface protein of the cells is directly contacted with the modified barcode, and the work time is generally shorter than that of the USB method (2 hours of processing). The shorter work time reduces the impact on the cells. The number of centrifugation steps is reduced, so cell loss during work can be reduced.

[0026] 5. Kit (2) The present invention also relates in one aspect to a kit for use in the method for labeling cells with a barcode, including, but not limited to, a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell. In one embodiment, the kit further comprises: direct sign The method can be used in "6. Method for multiplexing analysis of a cell sample (2)" or "6. Method for multiplexing analysis of a cell sample (2)." "Method for labeling cells with barcodes" and "Barcodes modified with compounds that bind to amino groups on cell surface proteins" are described in "4. Method for labeling cells with barcodes". direct sign This is as explained in "How to do this (2)". In one embodiment, the compound that binds to amino groups on cell surface proteins of the cells is sulfo-N-hydroxysuccinimide. In addition, unless there are any particular technical problems, the contents explained in "2. Kit (1)" also apply to Kit (2) in this section.

[0027] 6. Multiplexed Analysis Method for Cell Samples (2) The present invention further relates to a method for multiplexed analysis of a cell sample, including, but not limited to, the steps of: (1A) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (1B) contacting a single cell with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell; (2) Mixing all or a portion of the barcode-labeled cell population or single cells of (1); and, (3) Analyze the mixture of cells from (2). The method includes: (1A) and (1B) are the steps in "4. Barcoding cells" direct sign This is as explained in "How to do this (2)". (2) and (3) are as described in “3. Multiplexed analysis method for cell samples (1)”. In one embodiment, the analysis is cellular RNA analysis. The method may be in vivo, ex vivo or in vitro, preferably an in vitro method. In addition, unless there are any particular technical problems, the contents explained in "3. Multiplexed analysis method for cell samples (1)" also apply to the multiplexed analysis method for cell samples (2) in this section. EXAMPLES

[0028] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0029] Materials and Methods Unless otherwise stated, the following materials and methods were used in the examples.

[0030] 1.Cell culture R1 ES cells were provided by Dr. Sado (Kindai University). EB3 ES cells (AES0139) (Non-Patent Documents 15, 16) were obtained from the Cell Bank of the RIKEN BioResource Research Center. Undifferentiated ES cells were maintained with minor modifications according to the method described in Non-Patent Document 17. Briefly, ES cells were cultured on mitomycin C-treated mouse embryonic fibroblast feeder cells in Glasgow Minimum Essential Medium (GMEM; Merck, G5154) supplemented with 14% Knockout Serum Replacement (KSR; Thermo Fisher Scientific™, 10828028), 1% fetal bovine serum (FBS), 1,000 U / mL leukemia inhibitory factor (LIF), 0.11 mg / mL sodium pyruvate (Natalai Tesque, 29806-12), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific™, 21985023), 1× non-essential amino acids (Thermo Fisher Scientific™, 11140076) and 1× GlutaMAX supplement (Thermo Fisher Scientific™, 350061). For subculture, 0.25% trypsin (Fujifilm, 201-16945) was used.

[0031] 2. Differentiation of ES cells After removing the feeder cells, 1 × 10 4 ES cells were cultured in a low cell attachment U-bottom 96-well plate ( NunclonSphera 96-Well U-Shaped-Bottom Microplates (Thermo Fisher Scientific™, 174925) were seeded in 100 μL of differentiation medium (GMEM supplemented with 15% FBS, 0.11 mg / mL sodium pyruvate, 0.1 mM 2-mercaptoethanol, 1× non-essential amino acids, and 1× GlutaMAX supplement) and cultured for 24 hours. The spheroids generated in the plates were transferred to low cell attachment 90 mm culture dishes (Nunclon Sphera 90 mm dishes, 174945) and cultured for an additional 4 days to form embryoid bodies (EBs). The EBs were washed three times with PBS, dissociated into single cells using 0.25% trypsin, and the cells were seeded on gelatin-treated culture dishes and cultured for 7 days to induce further differentiation.

[0032] 3. Preparation of Single Cells for scRNA-seq Analysis ES cells were dispersed into single cells and cultured on a gelatin-treated culture dish for 1 hour, and then the feeder cells were removed. The floating cells were collected as undifferentiated ES cells. The differentiated ES cells were washed three times with PBS, treated with 0.25% trypsin, and dispersed into single cells. Dead cells in the cell suspension were removed by Percoll centrifugation. Briefly, the cells were cultured in RPMI 1640 (Fuji Film, 183-02165) supplemented with 5% FBS and 10 mM HEPES (Natalai Tesque, 17557-94). 25% adjusted concentration The cells were suspended in Percoll PLUS (Cytiva, 17544501) solution and layered on a 65% Percoll PLUS solution. After centrifugation at 1,000g for 20 minutes, the interphase was collected and washed with differentiation medium. The cell suspension was filtered through a 40 μm cell strainer, and the percentage of trypan blue-unstained cells was measured as the cell viability.

[0033] 4.USB sign S-NHS-biotin stock solution was prepared by dissolving EZ-Link Sulfo-NHS-biotin (Thermo Fisher Scientific™, 21217) in PBS at a concentration of 10 mg / mL and stored in aliquots at −80° C. until use. 0.5-2 x 10 cells prepared in "3. Preparation of single cells for scRNA-seq analysis" 6 Single Cells Containing 1 Single Cell of, The cells were washed once with ice-cold PBS supplemented with 1% FBS. The washed cells were suspended in 500 μL of ice-cold S-NHS-biotin working solution in PBS supplemented with 1% FBS and incubated on ice for 10 minutes. The S-NHS-biotin concentration in the examples was 10 μg / mL. After incubation on ice, 3 mL of ice-cold PBS containing 3% FBS was added to the reaction, and the cells were centrifuged at 300 g for 5 min at 4° C. The cells were further washed twice with Cell Staining Buffer (BioLegend, 420201) and then kept on ice until the next step of barcode oligo DNA binding.

[0034] 5. Antibody labeling with anti-CDH1 antibody (control experiment) 0.5-2 x 10 cells prepared in "3. Preparation of single cells for scRNA-seq analysis" 6 Single cells containing 1 single cell were washed once with ice-cold Cell Staining Buffer, suspended in 50 μL of ice-cold blocking solution (0.05 μg / mL TruStain FcX PLUS antibody [BioLegend, 156603] in Cell Staining Buffer) and incubated on ice for 10 min. Ice-cold primary antibody solution (0.05 μg / mL biotinylated CDC324 [E-cadherin] rat monoclonal antibody [Thermo Fisher Scientific™, 13-3249-82] in Cell Staining Buffer) was mixed with the cell suspension and the cells were incubated on ice for 30 min. Then, the cells were washed three times with ice-cold Cell Staining Buffer and kept on ice until the barcode DNA was bound. As an isotype control, a biotinylated rat IgG1 isotype control (Thermo Fisher Scientific™, 13-4301-82) was used.

[0035] 6. Barcode Oligo DNA Binding Using five types of TotalSeq® PE streptavidin (BioLegend, 405251, 405253, 405255, 405257, 405259) or TotalSeq® anti-biotin antibody (BioLegend, 409008), barcode oligo DNA was bound to the biotin-labeled cells prepared in "4. USB labeling" or the antibody-labeled cells prepared in "5. Antibody labeling with anti-CDH1 antibody (control experiment)" as follows. Information on the barcode oligo DNA used is shown in the table below. [Table 1] Cells were suspended in 100 μL of ice-cold 0.6 μg / mL TotalSeq® PE streptavidin or 2 μg / mL TotalSeq® anti-biotin antibody, diluted in Cell Staining Buffer, incubated on ice for 30 min, and then washed three times. Cell suspensions were filtered through a 40 μm cell strainer and equal numbers of cells from each sample were combined into a single tube and used for cell capture and cDNA synthesis using a BD Rhapsody™ instrument.

[0036] 7. Flow Cytometry First, single cell suspensions of undifferentiated ES cells and differentiated ES cells were prepared by the same method as in 1-3 above. Specifically, normal F344 rat lungs were cut into 0.5 mm pieces using a razor blade. 2The tissue was then chopped into small pieces and digested in Liberase solution [RPMI-1640 (Natalai Tesque)] supplemented with 10% FBS, 10 mM HEPES pH 7.2-7.4, 0.25 mg / ml Liberase™ (Roche), and 2000 U / mL DNase I (Merck KGaA) for 60 min at 37°C to prepare single cell suspensions. Dead cells were removed with Percoll. Alternatively, growth plate cartilage from the proximal tibia was collected from 3-week-old mice and minced into 1-2 mm pieces. It was then dispersed into single cells (groups of single cells) in Liberse™ (Roche) solution with constant shaking for 120-210 min. Reagents for cell staining included biotinylated CDC324 (E-cadherin) rat monoclonal antibody (Thermo Fisher Scientific™, 13-3249-82), TotalSeq® PE streptavidin (BioLegend, 405251), PerCP-Cy5.5 anti-rat CD45 (Bio-Rad, clone OX-1), PE anti-rat CD31 (Bio-Rad, clone TLD-3A12), streptavidin-APC (BioLegend, 405207), APC rat anti-mouse CD45 (BD Biosciences , 559864) and PE anti-mouse H-2 antibody (BioLegend, Inc. 125505). Stained cells were analyzed by flow cytometry using an EC800 Cell Analyzer (Sony), a CytoFLEX flow cytometer (Beckman Coulter), or a FACS Arria II flow cytometer (BD Biosciences). All animal experiments were reviewed and approved by the Kyoto University Institutional Animal Committee and the Tokyo University of Science Animal Experimentation Committee (approval numbers: S17034, S18029, S19024, S20019).

[0037] 8. Immunofluorescence Cells grown on gelatin-treated culture dishes were fixed with 4% paraformaldehyde in PBS overnight at 4°C, then washed three times with washing buffer (PBS containing 0.1% Triton X-100). Cells were incubated in blocking buffer (PBS containing 1% BSA and 0.1% Triton X-100) and then incubated overnight with primary antibodies diluted in blocking buffer. After washing with washing buffer, cells were incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature, then mounted with 2% DABCO (Sigma, D2522) in PBS containing 40% glycerol. Images were taken using a LEICA AF6500 fluorescence imaging system (Leica). The antibodies and dilution rates used in the examples were as follows. Primary antibodies-biotinylated CDC324 (E-cadherin: CDH1) rat monoclonal antibody (Thermo Fisher Scientific™, 13-3249-82) (1:200) and anti-OCT4 rabbit polyclonal antibody (Abcam, ab19857) (1:400); Secondary antibodies-Alexa Fluor 488 anti-rat donkey IgG (Thermo Fisher Scientific™, A21208) (1:500) and Alexa Fluor 555 anti-rabbit donkey IgG (Thermo Fisher Scientific™, A31572) (1:500).

[0038] 9. Cell viability test Undifferentiated ES cells were treated with 100 μg / mL S-NHS-biotin (10 times the concentration used for multiplex scRNA-seq) and TotalSeq® PE streptavidin, and 3 × 10 3 Cells were seeded in gelatin-treated 3.5 cm culture dishes. In parallel, the same number of untreated cells were seeded. After 5 days of culture, cells were stained with hematoxylin to examine the colony formation efficiency of untreated controls and treated samples.

[0039] 10.scRNA-seq scRNA-seq library of For the preparation, we used the BD Rhapsody Express Single-cell Analysis system (BD Biosciences) and parts of the Targeted mRNA and AbSeq Amplification Kit (BD Biosciences, 633771). Cells labeled with barcode oligo DNA (1.6 × 10 4 ) were captured in a microwell cartridge, and cell capture beads bound with single-cell-derived mRNA and barcode oligo DNA were collected and used for cDNA synthesis. cDNA synthesis was performed according to the BD Rhapsody Express manual. cDNA and barcode oligo DNA were amplified according to the TAS-seq protocol (Non-Patent Document 10). Briefly, poly-C tails were added to the cell capture beads to which cDNA and barcode oligo DNA were bound by treating with 0.75 U / μL terminal deoxynucleotidyl transferase (TdT; Enzymatics, P7070L) solution (containing 1 mM dCTP [GE Healthcare, 28406512] and 0.05 mM ddCTP [GE Healthcare, 27206101] in 1×TdT buffer [Thermo Fisher Scientific™, 16314015]) for 30 minutes while mixing at 1,200 rpm. To synthesize second strand cDNA sequences, poly-C-tailed cDNA beads were treated with 1x KAPA HiFi HS ReadyMix (Roche, 7958935001) using the 5'-BDWTAv2-9G primer in a program of [98°C for 20 seconds, 47°C for 1 minute, 72°C for 2 minutes] x 16 cycles. Next, 1x KAPA HiFi HS ReadyMix with three primers (5'-BDWTAv2, Universal Oligo-long, and TotalSeq-ADT-oligo 1) was added to the reaction, and the whole transcriptome was amplified using a program of [98°C for 20 s, 63°C for 20 s, 72°C for 5 min] x 7 cycles. The amplified cDNA and barcode oligo DNA were separated by size selection using AMPure XP beads (Beckman Coulter, A63881). The cDNA was further treated with 1× KAPA HiFi HS ReadyMix using two primers (5′-BDWTAv2 and Universal Oligo-long) with a program of [98°C for 20 sec, 65°C for 20 sec, 72°C for 5 min] × 5 cycles. The barcode oligo DNA was further amplified with two primers (TotalSeq-ADT-oligo 2 and Universal Oligo-long) with a program of [98°C for 20 sec, 65°C for 20 sec, 72°C for 5 min] × 12 cycles. The primer sequences are as shown in the table below. [Table 2] The 5' ends of SEQ ID NOs: 8 and 9 are 2 -C 12 are bonded. The amplified cDNA and barcode oligo DNA sequences were purified using AMPure XP beads, and the size distribution and yield were checked by a bioanalyzer (Agilent) using a High Sensitivity DNA Kit (Agilent, 5067-4626). Sequence data were collected at ImmunoGeneTeqs, Inc. using a NovaSeq 6000 S4 flow cell (Illumina).

[0040] 11. Data Analysis The demultiplexed and DBEC processed sequence data were imported into R (4.0.2) and cells determined to be “doublets” or “not detected” from the tag count analysis and cells with low sequence reads were removed. Genes with low read counts or genes that were not found in a sufficient number of cells were also removed. The Seurat package (4.0.5) was used for the subsequent analysis and parameters were modified according to the tutorial workflow (Non-Patent Document 18). The tutorial workflow is typically as follows: “Create a Seurat object using an expression table. After quality checking the data, normalize and extract feature genes. Dimension reduction is performed to form clusters of cells with the same cell type and state. Classify and label the cells in the dataset by cluster analysis. Identify marker genes and differentially expressed genes for each cluster to determine and annotate cell types.” In this example, cells with more than 10% mitochondrial genes were excluded. Next, the count matrix was used to perform the default global scaling normalization method LogNormalize. The extraction method used was vst. In "vst", essentially, genes with high variance of expression levels are detected relative to the average expression level obtained across all cells. First, a line is fitted to the relationship between log(variance) and log(mean) using local polynomial regression (loess). This allows for feature standardization without removing more than expected variance. Next, the features are standardized using the observed mean and expected variance (given by the fitted line). After clipping to the maximum value, the variance of the features is calculated for the standardized values. To reduce the influence of technical outliers, the standardized values ​​are truncated at the maximum value. The top 2,000 highly variable genes were calculated using FindVariableFeatures (a command that identifies features (e.g., genes) that are outliers on the "mean variation plot"). After scaling the data, PCA was performed. Based on the Euclidean distance in the space occupied by the first 30 principal components identified, a K-nearest neighbor (KNN) graph was constructed and the Louvain algorithm was applied to identify clusters. The initial clustering yielded 11 clusters. In addition, UMAP was performed using 2,000 highly variable genes and 30 principal components, resulting in 11 subclusters. Marker genes for each cluster were identified using FindAllMarker (a command to identify enriched genes in each cluster formed; in this example, the Wilcoxon Rank Sum test is applied by default) (min.pct=0.25, logfc.threshold=0.25, set.only.pos=TRUE). Expression of the E-cadherin gene (Cdh1) was plotted with FeaturePlot (a command to visualize "features" on a dimensionality reduction plot).

[0041] 12. Data Availability All raw FASTQ sequence files were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers DRR333192-DRR333193. Working Example

[0042] Example 1 Cell labeling by USB method 1.1 Principle of cell labeling The present inventors developed a method called S-NHS- Biotin We developed a new cell labeling technique for multiplexed scRNA-seq using the USB method. While the conventional method (Ab method) targets specific proteins on the cell surface, the USB method targets amine groups present in almost all proteins, making it a universal method that does not require specific surface antigens. The conventional method cannot label cells if the target surface protein is not expressed, but the USB method can label cells as long as the surface protein is present (Figure 1). In this example, as a proof-of-concept experiment, mouse ES cells were used to compare two analytical methods for cells in an undifferentiated state and differentiated cells. In mouse ES cells, almost all cells express the adhesion molecule E-cadherin (CDH1) (Non-Patent Document 9) in an undifferentiated state, but the number of cells that do not express CDH1 increases with differentiation (Figure 6). Thus, antibody assays using anti-CDH1 antibodies label most cells in an undifferentiated state, but not all cells in differentiated cells. In contrast, the USB method does not depend on CDH1 expression, and is therefore predicted to be able to label cells in both undifferentiated and differentiated states. 1.2 Cell labeling with USB Single cells were prepared from undifferentiated mouse ES cells (R1 line) and cells differentiated from R1 cells by embryoid body formation, and used in the following experiments. The cell sample was divided into two halves, one half was treated with the Ab method, i.e., biotin-conjugated anti-CDH1 antibody, and the other half was biotinylated to cell surface proteins using the USB method. The barcode DNA-labeled streptavidin used was conjugated to the fluorescent dye phycoerythrin (PE), so the labeling efficiency was examined by flow cytometry analysis (Figure 2). Using antibody labeling, the percentage of PE-positive cells in undifferentiated ES cells was 87.8%, whereas the percentage of PE-positive cells in differentiated ES cells was approximately 10%. Immunostaining with anti-CDH1 antibody showed that almost all cells in the undifferentiated ES cell sample were CDH1-positive (Fig. 3), whereas CDH1 expression was detected only in a portion of the differentiated ES cells, which was consistent with the results of flow cytometry. In contrast, when the USB method was used, the percentages of PE-positive cells in the undifferentiated ES cell and differentiated cell samples were 99.6% and 96.4%, respectively (Figures 2 and 3). Fluorescence microscopy also showed that almost all cells were fluorescently labeled (Figure 3, top). These results indicate that the USB method can efficiently label most cells in both the undifferentiated and differentiated states.

[0043] Example 2 USB method operation and cell survival In this example, it was demonstrated that the USB method procedure does not affect cell survival. The series of experimental procedures using the USB method may impair the condition and proliferation of cells. To examine this possibility, ES cells were treated with the USB method, seeded on a culture dish, and colony formation ability was measured (Figure 7). As a result, no difference in colony formation rate was observed between the unlabeled control group and the USB group, even when the cells were treated with S-NHS-biotin at a concentration 10 times higher than the concentration used in the USB method (0.1 mg / mL). Therefore, we concluded that the USB method does not change cell viability.

[0044] Example 3: Example of sample multiplexing and scRNA-seq analysis using the USB method The USB method of the present invention can comprehensively bind DNA barcodes to most of various cells through biotin-streptavidin binding, and the experimental manipulation does not impair the viability of the cells. In this example, we investigated whether the USB technology can be used for multiplexing in scRNA-Seq analysis and whether the expression profile of cells is affected by the experimental manipulation. Undifferentiated and differentiated R1 ES cells were treated with either the USB method or the Ab method. In addition, undifferentiated and differentiated cells of another ES cell line, EB3, were labeled using the USB method. These six samples were mixed in equal amounts, and approximately 16,000 single cells were captured using the BD Rhapsody™ system. A portion of these cells (~4,000 cells) were used for cDNA synthesis and amplification for whole transcriptome analysis, as well as amplification of barcode oligo DNA tags, according to the TAS-seq protocol (Non-Patent Document 10). The size distribution and yield of the amplified cDNA and barcode oligo DNA were estimated, and appropriate amplification was confirmed (Figure 8). After removing dead cells, doublet cells, and cells for which tags could not be identified, the remaining 2,089 cells were subjected to informatics analysis (Table 3). [Table 3] Data obtained from the undifferentiated and differentiated states of the R1 ES cell line labeled by either USB or Ab methods are shown in Figure 4. Using the uniform manifold approximation and projection (UMAP) method, the cells were classified into 11 clusters. Based on the expressed genes, clusters 0, 1, and 3 corresponded to pluripotent cells, naive cells, and primed pluripotent cells, respectively, and were considered to be cells in an undifferentiated state (Figure 4, Table 4). [Table 4] It is noteworthy that the cells in these clusters labeled by the two different methods were classified into the same cluster (Figure 9). That is, the expression profile of the cells labeled by the USB method of the present invention was very similar to that of the cells labeled by the conventional Ab method. Therefore, it was revealed that the influence of the experimental operation by the USB method on not only cell growth but also the gene expression profile of each cell was negligible. Clusters other than 0, 1, and 3 were identified as differentiated cells, including vascular smooth muscle cells (cluster 2), dopamine neurons (cluster 4), epithelial cells (cluster 5), and nerve cells (cluster 7), as well as cell clusters thought to be macrophages (cluster 8), glial cells (cluster 9), and vascular endothelial cells (cluster 10). Mouse embryonic fibroblast (MEF) feeder cells, which could not be completely removed, were detected as cluster 6. Analysis of cell samples from each cluster revealed that USB-labeled cells were present in all clusters (Figures 4 and 9), demonstrating that this method can indeed capture various types of differentiated cells. In differentiated cell clusters 2, 4, and 7, the number of cells labeled by the Ab method was decreased compared to the cells labeled by the UBS method (Fig. 4, Fig. 9). This is considered to be due to the suppression of Chd1 gene expression in these differentiated cells, which led to a decrease in the labeling efficiency by the Ab method using CDH1 antibody. In the Cdh1-positive clusters (clusters 0, 1, 3, and 5), the number of cells detected was almost the same between the samples labeled by the USB method and the Ab method. However, in the Cdh1-negative clusters (clusters 2, 4, 6, 7, 8, 9, and 10), the number of cells labeled by the Ab method was obviously less than that by the USB method (Fig. 5), indicating the limitation of the Ab method for detecting differentiated cells. [Table 5] This tendency was also observed with the EB3 line, indicating that the USB method can be applied to different ES cell lines (Figure 10). CDH1-negative cells are not considered to be labeled by the anti-CDH1 antibody and should not be detected. However, in the antibody-treated cells, Cdh1-negative cells were still present, although their numbers were clearly reduced. teeth The barcode oligo DNA was definitely included (Table 5, Figures 4 and 5). One possible cause is non-specific binding of the antibody or streptavidin to the cells. Indeed, we confirmed that the barcode oligo DNA was amplified in both streptavidin-only and isotype control antibody / streptavidin-treated samples (Figures 13 and 14). Since the barcode oligo DNA was not amplified at all in the unlabeled samples, the amplification observed in the streptavidin-only and isotype control antibody-treated samples (Figure 14, arrows) is likely due to non-specific binding of streptavidin and / or isotype control antibody to the cells. Since the amplification by scRNA-seq is considered to be more sensitive than flow cytometry, some cells that do not express CDH1 (or express very low amounts) may have been classified as positive cells due to nonspecific binding by streptavidin and / or isotype control antibodies. However, in cell labeling in scRNA-seq multiplexing, it is very important that all cells in the sample are labeled uniformly, and a small amount of nonspecific binding in the Ab method itself is not considered to affect the results of multiplexing.

[0045] Example 4 Sulfo-NHS-esterification of barcode oligo DNA In this example, the barcode oligo DNA was esterified with sulfo-NHS. The materials used are as follows: - Oligo DNA modified with -COOH at the 5' end; For cell labeling validation experiments, 3'-end 6-FAM modification was also performed (6-FAM modification is not required for sample multiplexing in scRNA-seq). EDC (1-ethyl-3-[3-dimethylamino Propyl ]Carbodiimide hydrochloride) (Thermo Fisher SCIENTIFIC(trademark) , A35391(10x1mg, No-Weigh(TM) Format); Sulfo-NHS (Thermo Fisher SCIENTIFIC(trademark) ,A39269(10x2mg,No-Weigh(TM) Format); ·Activation buffer (0.1M MES, 0.5M NaCl, pH6.0); ·PD MidiTrap G-25(Cytiva, 28918008); and Nanosep Centrifugal Filtration Device, molecular weight cutoff 3K (Nihon Genetics, OD003C33) The method used in this example is as follows. (4-i) Load 918 μL of activation buffer into the reaction tube; (4-ii) Dissolve 1 mg of EDC in 100 μL of activation buffer, and add 40 μL to a reaction tube; (4-iii) Add 2 nmol (20 μL) of oligo DNA to the reaction tube; (4-iv) Dissolve 2 mg of sulfo-NHS in 40 μL of activation buffer and add 22 μL to a reaction tube; (4-v) Leave at room temperature for 15 minutes; (The following operations are carried out at 4℃ or on ice.) (4-vi) Remove unreacted reagents using PD MidiTrap G-25; (4-vii) The buffer solution is replaced with PBS using a Nanosep centrifugal filtration device (buffer replacement by ultrafiltration). This procedure is repeated twice to concentrate the sulfo-NHS-esterified oligo-DNA (NEO). The sulfo-NHS-esterified oligo-DNA obtained above was stored at -80°C until use in the following examples.

[0046] Example 5 Verification of cell labeling efficiency with sulfo-NHS-esterified oligo-DNA (NEO) In this example, cells were labeled with sulfo-NHS-esterified oligo-DNA (NEO) (single-step method) and the labeling efficiency was verified. All operations and reactions in this example were carried out at 4°C or on ice. (5-i) 1x10 cells dispersed into single cells by the method described in "3. Preparation of single cells for scRNA-seq analysis" in "Materials and Methods". 6 Prepare the following. (5-ii) 4 ng of sulfo-NHS-esterified oligo DNA (NEO) in 20 μL of 0.1% BSA / PBS in Dilute. (5-iii) The dispersed single cells prepared in (5-i) are suspended in the NEO solution prepared in (5-ii) and treated on ice for 20 minutes. (5-iv) Add 3 mL of 3% FBS / PBS and centrifuge (300 x g, 10 minutes). (5-v) Remove the supernatant, suspend the cells in 1 mL of Cell Staining Buffer (Catalog #420201) (Biolegend), and centrifuge (300 x g, 10 minutes). Repeat this procedure three times. The labeled cells described above were used for single-cell RNA-seq analysis (scRNA-seq analysis). Furthermore, the cell labeling efficiency was verified as follows. The cell suspension labeled with 6-FAM-modified sulfo-NHS-esterified oligo-DNA (NEO) was passed through a cell strainer and then subjected to flow cytometry analysis. The results are shown in FIG. 15. Compared to the labeled control (labeling rate 0.01%), the single-step labeling method in this example resulted in almost all cells ( about It was confirmed that sufficient labeling was achieved with a 99.6% success rate.

[0047] Example 6 Multiplex scRNA-seq analysis of various cells labeled with the single-step method Undifferentiated ES cells cultured in naive pluripotent stem cell maintenance medium (2i medium), undifferentiated ES cells cultured in normal ES cell medium (Normal ESC medium), differentiated ES cells cultured in LIF(-) serum medium for 5 days (Day5 differentiated), and differentiated ES cells cultured in LIF(-) serum medium for 10 days (Day10 differentiated) were labeled with different barcode tags using a single-step method and then subjected to multiple scRNA-seq analysis (Figure 16). Nine cell types were detected by UMAP clustering. The nine cell types and their marker genes (top 20 representative ones) are as follows: [Table 6] The results are shown in Figure 17 and Table 7. [Table 7] In 2i medium, many naive pluripotent cells were identified, while in normal ESC medium, primed pluripotent cells were the most abundant. In differentiated cells on day 5, cardiovascular cells dominated, and in differentiated cells on day 10, differentiated cells, mainly vascular smooth muscle cells, were detected. [Sequence table]

Claims

1. A method for labeling any type of cell with a barcode, comprising: (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins of single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; and, (ii) contacting each cell group or each single cell with a barcode-labeled biotin-binding substance; The method includes:

2. (ia) biotinylating a cell surface protein for a cell population including a plurality of single cells; and, (ii) Contacting each cell group with a barcode-labeled biotin-binding substance The method of claim 1 , comprising:

3. The method according to claim 1 or 2, wherein in (i) an amino group, a sulfhydryl group or a carboxyl group of a cell surface protein is biotinylated.

4. In (i), the reagent used for biotinylation is selected from the group consisting of sulfo-N-hydroxysuccinimide-biotin, N-hydroxysuccinimide-biotin, and pentafluorophenyl-biotin; wherein the bond between biotin and sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, or pentafluorophenyl may include a spacer; The method according to any one of claims 1 to 3.

5. The method according to claim 4, wherein the reagent used for biotinylation in (i) has been frozen and stored.

6. The method according to any one of claims 1 to 5, wherein the biotin-binding substance is selected from the group consisting of streptavidin, avidin and an anti-biotin antibody.

7. The method of claim 6 , wherein the biotin-binding substance is streptavidin.

8. The method according to any one of claims 1 to 7, wherein the barcode is an oligo-DNA.

9. The method according to any one of claims 1 to 8, wherein live cells are labeled.

10. The method according to any one of claims 1 to 8, wherein fixed cells are labeled.

11. a reagent for biotinylating cell surface proteins of a cell; and Barcoded biotin-binding substances A kit for use in the method for labeling any type of cell with a barcode according to any one of claims 1 to 10, comprising:

12. The kit of claim 11, wherein the reagent for biotinylating cell surface proteins of the cells is sulfo-N-hydroxysuccinimide-biotin.

13. A method for multiplexed analysis of a cell sample of any type of cell, comprising: (ia) for a cell population including a plurality of single cells, biotinylating a cell surface protein of the cells; (ib) biotinylating cell surface proteins of single cells; or (ic) biotinylating cell surface proteins of a plurality of cells, and then separating the biotinylated cells into a cell population comprising a plurality of single cells or into single cells; (ii) contacting each group of cells or each single cell with a barcode-labeled biotin-binding substance; (iii) mixing all or a portion of the barcode-labeled cell population or single cells of (ii); and, (iv) analyzing the mixture of cells of (iii); The method includes:

14. (ia) biotinylating a cell surface protein for a cell population including a plurality of single cells; and, (ii) Contacting each cell group with a barcode-labeled biotin-binding substance Including, The method of claim 13.

15. The method of claim 13 or 14, wherein the analysis is cellular RNA analysis.

16. A method for labeling any type of cell with a barcode, comprising the steps of: (1) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group, a sulfhydryl group, or a carboxyl group of a cell surface protein of the cells; or (2) contacting a single cell with a barcode modified with a compound that binds to amino, sulfhydryl, or carboxyl groups of cell surface proteins of the cell; The method includes:

17. A method for labeling any type of cell with a barcode, comprising: (1) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (2) contacting the single cell with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell; The method includes:

18. 18. The method of claim 17, wherein the compound that binds to amino groups of cell surface proteins of cells is selected from the group consisting of sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, and pentafluorophenyl.

19. The method according to claim 17 or 18, wherein the compound that binds to an amino group of a cell surface protein of a cell has been cryopreserved.

20. The method according to any one of claims 16 to 19, wherein the barcode is an oligo-DNA.

21. The method according to any one of claims 16 to 20, wherein live cells are labeled.

22. The method according to any one of claims 16 to 20, wherein fixed cells are labeled.

23. A kit for use in a method for labeling any type of cell with a barcode as described in any one of claims 16, 20-22, comprising a barcode modified with a compound that binds to an amino group, a sulfhydryl group or a carboxyl group of a cell surface protein of the cell.

24. A kit for use in the method for labeling any type of cell with a barcode according to any one of claims 17 to 22, comprising a barcode modified with a compound that binds to the amino group of a cell surface protein of the cell.

25. The kit according to claim 24, wherein the compound that binds to amino groups of cell surface proteins of cells is sulfo-N-hydroxysuccinimide.

26. A method for multiplexed analysis of a cell sample of any type of cell, comprising: (1A) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group, a sulfhydryl group, or a carboxyl group of a cell surface protein of the cells; or (1B) contacting a single cell with a barcode modified with a compound that binds to an amino group, a sulfhydryl group, or a carboxyl group of a cell surface protein of the cell; (2) Mixing all or a part of the barcode-labeled cell population or single cells of (1); and, (3) Analyzing the mixture of cells (2) The method includes:

27. A method for multiplexed analysis of a cell sample of any type of cell, comprising: (1A) contacting a population of cells including a plurality of single cells with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cells; or (1B) contacting a single cell with a barcode modified with a compound that binds to an amino group of a cell surface protein of the cell; (2) Mixing all or a part of the barcode-labeled cell population or single cells of (1); and, (3) Analyzing the mixture of cells (2) The method includes:

28. 28. The method of claim 26 or 27, wherein the analysis is cellular RNA analysis.

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