Oligonucleotide and method for marking cells

Abstract

The invention provides oligonucleotide and method for marking cells, the nucleotide sequence of the oligonucleotide is shown as SEQ ID NO.1, the oligonucleotide sequentially comprises a primer 1, a bar code, a primer 2, a Poly A tail and a tail adding signal from the 5'end to the 3 'end, the nucleotide sequences of the primer 1 and the primer 2 are sequentially shown as SEQ ID NO.2-SEQ ID NO.3, and the bar code is composed of 10 random basic groups. According to the present invention, the primer 1 and the primer 2 in the oligonucleotide for labeling the cells do not match the sequence in the human genome, such that the library expression condition can be simply and rapidly verified through the PCR, the cells are labeled in batch during the in-vitro culture so as to provide the unique RNA bar code for each cell, such that the cells can be tracked, and the detection result is accurate. In addition, a single cell transcriptome sequencing technology is particularly optimized, so that the single cell transcriptome can be accurately detected in subsequent single cell sequencing.

Description

Technical Field

[0001] The present invention relates to the field of single-cell sequencing technology, and in particular to a transcriptome expression sequence tag method for batch labeling and dynamic lineage tracking of cells cultured in vitro. Background Art

[0002] Cell heterogeneity refers to the presence of different types of cells in the same source tissue, which differ in morphology, function, and gene expression. Taking tumor cell heterogeneity as an example, this heterogeneity may lead to poor treatment effects of tumors because different types of tumor cells have different sensitivities to therapeutic drugs, and may even lead to tumor recurrence and metastasis. The development of single-cell sequencing technology allows researchers to have a deeper understanding of the characteristics and functions of different cell types in the same tissue. Through single-cell sequencing, subpopulations in cell populations can be revealed and their differences in gene expression levels can be analyzed. Researchers focus on the evolution and mutation process of tumor cells, explore the relationship between different tumor cell types, and how they are formed and evolved. This helps to understand the source of tumor heterogeneity and provide a basis for the development of more effective treatment strategies. Currently, 10x Genomics' Chromium single-cell sequencing platform and InDrop's Drop-seq single-cell sequencing platform use microfluidic chip technology to mark cells and sort cells according to DNA barcode sequences after sequencing. However, such marking is cross-sectional and cannot track the fate of its offspring. To achieve sequencing at different time points to track the differentiation and migration of cells, it is necessary to use mathematical models and computational algorithms to infer the lineage relationship of tumor cells. Common methods include constructing evolutionary trees, analyzing mutation co-occurrence patterns, and calculating cell state transition probabilities.

[0003] Therefore, it is necessary to develop a method that can track cells quickly and conveniently. Summary of the invention

[0004] The purpose of the present invention is to provide an oligonucleotide and method for labeling cells, so that cells can be labeled in batches during in vitro culture so that each cell has a unique RNA barcode, thereby tracking the cells, and specifically optimizing the single-cell transcriptome sequencing technology so that it can be accurately measured in subsequent single-cell sequencing.

[0005] The present invention adopts the following technical solution:

[0006] In the first aspect of the present invention, an oligonucleotide for labeling cells is provided, the nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.1, and the oligonucleotide comprises, from the 5' end to the 3' end:

[0007] Primer 1, barcode, primer 2, Poly A tail and tailing signal, the nucleotide sequences of primer 1 and primer 2 are shown in SEQ ID NO.2-SEQ ID NO.3 respectively, and the barcode consists of 10 random bases.

[0008] In the second aspect of the present invention, an expression vector for labeling cells is provided, wherein the expression vector contains the oligonucleotide for labeling cells.

[0009] In the third aspect of the present invention, a method for constructing the expression vector for labeling cells is provided, the method comprising:

[0010] Synthetically obtaining the oligonucleotides for labeling cells;

[0011] amplifying the oligonucleotide by PCR to obtain a PCR product;

[0012] The PCR product is connected to an expression vector to obtain an expression vector for labeling cells.

[0013] In a fourth aspect of the present invention, a method for labeling cells is provided, the method comprising:

[0014] The expression vector for labeling cells is transfected into cells cultured in vitro.

[0015] In a fifth aspect of the present invention, a method for constructing a high-throughput single-cell full-length transcriptome sequencing library is provided, the method comprising:

[0016] First, the cells are labeled using the method described above to obtain labeled single cells;

[0017] The labeled single cells are sequentially lysed to extract RNA, the RNA is transcribed into cDNA using a reverse transcriptase, and a library is constructed for the cDNA to obtain a single-cell transcriptome sequencing library that can be directly used for subsequent sequencing.

[0018] In a sixth aspect of the present invention, a high-throughput single-cell full-length transcriptome sequencing library prepared by the method is provided.

[0019] In the seventh aspect of the present invention, the application of the high-throughput single-cell full-length transcriptome sequencing library in single-cell sequencing is provided.

[0020] In the eighth aspect of the present invention, the application of the high-throughput single-cell full-length transcriptome sequencing library in single-cell sequencing is provided.

[0021] In the ninth aspect of the present invention, a high-throughput single-cell sequencing method is provided, the method comprising using the high-throughput single-cell full-length transcriptome sequencing library, and then using a high-throughput sequencing platform for sequencing.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. The oligonucleotides and methods for labeling cells provided by the present invention can be normally expressed in human in vitro cultured cells, and the products have no corresponding translated polypeptides or proteins, and have no effect on normal cell activities. Primers 1 and 2 in the oligonucleotides for labeling cells do not match the sequences in the human genome, so the expression of the library can be simply and quickly verified by PCR.

[0024] Compared with the prior art solutions, the present invention has the following advantages:

[0025] (1) Accurate cell tracking: Suitable for single-cell transcriptome sequencing with 3' end library construction, can be read by single-cell transcriptome sequencing, confirm the library through primer sequence, achieve uniqueness through barcode region, and track cell lineage changes through barcode combination.

[0026] (2) High-throughput labeling: 10-bit random sequence, theoretically can generate 4 10 = 1,048,576 unique barcodes. In transfection, higher labeling throughput can be achieved by combining barcodes.

[0027] (3) High expression efficiency and high read length coverage: The entire library is within 100 bp, with high expression efficiency, no sequence matching the human genome, no interference with the genome, and no expression of peptides and proteins. The full length is controlled within 100 bp, ensuring that the library sequence is captured in the subsequent transcriptome single-cell sequencing library construction.

[0028] (4) Simple operation and easy implementation: no tedious steps such as resistance screening are required, and there is no interference with the target experimental process.

[0029] 2. The combination of cell lineage tracing and expression tags has important application value in biological research. Through this technology, researchers can track the development process of specific cell populations or their behavior under physiological conditions. These tags can help scientists solve a variety of biological problems, including: Developmental biology: understand the origin and development process of different cell types, and reveal the mechanism of cell fate determination. Tissue regeneration and repair: study the response of specific cells after injury and their role in tissue repair. Tumorigenesis and metastasis: track the origin and metastasis process of tumor cells, and help clarify the heterogeneity and drug resistance mechanism of tumors. Neuroscience: reveal the connection mode of neurons and their role in cognitive and behavioral functions. Drug development: evaluate the effect of drugs on specific cell types or tissues, and optimize treatment strategies. Gene editing and gene therapy: determine the effect and safety of specific gene editing or gene therapy strategies at the cellular and tissue levels. In short, the application of cell lineage tracing expression tags provides a powerful tool for understanding the structure and function of complex cell networks in organisms, and promotes research progress and clinical application in many fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The gene designed for the embodiment of the present invention consists of primer 1, barcode, primer 2, Poly A tail and tailing signal.

[0032] Figure 2 This is the electrophoresis verification diagram of the library plasmid restriction enzyme digestion.

[0033] Figure 3 This is a validation image of the library by ordinary Sanger sequencing. The 10-bit random sequence is located by the primer sequence, and its uniqueness can be seen.

[0034] Figure 4 The figure shows the plasmid structure and sequence of the library.

[0035] Figure 5 Comparison of single-cell transcriptome sequencing analysis based on clustering and expression profile tags. For continuously cultured cell samples (P0, P4...), Figure 5 A: Conventional single-cell transcriptome sequencing currently uses cell expression profiles to cluster cells and can only analyze changes in the overall level within the same cluster. Figure 5B: After adding expression profile tags, specific single cells can be identified through random sequence barcodes, and changes in cells from the same lineage and changes in target genes can be observed at different time points. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0037] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0039] The present application will be described in detail below with reference to embodiments and experimental data.

[0040] Example 1. Oligonucleotides for labeling cells

[0041] As a typical implementation method provided by this application, Figure 1 As shown, an oligonucleotide for marking cells is provided, the nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.1, 5'-AAGCAGTGGTATCAACGCAGAGTNNNNNNNNNNCTAATACGACTCACT ATAGGGCAAAAAAAAAAAATAAA-3'.

[0042] The oligonucleotide comprises, from the 5' end to the 3' end:

[0043] Primer 1, barcode, primer 2, Poly A tail and tailing signal, the nucleotide sequences of primer 1 and primer 2 are shown in SEQ ID NO.2-SEQ ID NO.3 respectively, and the barcode consists of 10 random bases.

[0044] Example 2: Method for labeling cells

[0045] 1. Plasmid construction:

[0046] (1) As an implementation method of the present invention, the oligonucleotide for labeling cells with a restriction site (shown in SEQ ID NO.4) can be directly synthesized, and the restriction site is added on the basis of SEQ ID NO.1:

[0047] (5'-GGTACCAAGCAGTGGTATCAACGCAGAGTNNNNNNNNNNCTAATAC GACTCACTATAGGGCAAAAAAAAAAAATAAACTCGAG-3'). The bold part GGTACC is the KpnI restriction site, and CTCGAG is the XhoI restriction site.

[0048] Then, the pcDNA3.1 plasmid is subjected to restriction enzyme reaction, and the plasmid and the target DNA fragment are subjected to restriction enzyme digestion by selecting appropriate restriction endonucleases. Subsequently, gel electrophoresis is performed, and the target DNA fragment is cut therefrom and gel purified. The restriction enzyme sites specifically selected in the embodiment of the present invention are KpnI and XhoI.

[0049] The pcDNA3.1 plasmid that has been digested and purified is ligated with the target DNA fragment, and DNA ligase is usually used to connect the two into a complete recombinant plasmid.

[0050] (2) As another implementation of the embodiment of the present invention,

[0051] Directly synthesize the oligonucleotide for labeling cells (shown in SEQ ID NO.1),

[0052] Then, primers with restriction sites (as shown in SEQ ID NO.5-SEQ ID NO.6) were designed, and PCR reaction was performed on the oligonucleotide shown in SEQ ID NO.1 to obtain PCR products.

[0053] Restriction sites were added on the basis of primer 1 and primer 2. The specific primer sequences are as follows:

[0054] 5'-CTAATACGACTCACTATAGGGCAAAAAAAAAAAATAAACTCGAG-3' (SEQ ID NO. 5);

[0055] 5'-GGTACCAAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO. 6).

[0056] The synthesized oligonucleotide shown in SEQ ID NO.5 was amplified by PCR using the two designed primers to obtain the DNA fragment required for connection with pcDNA3.1.

[0057] (4) Enzyme digestion and gel purification:

[0058] The PCR product and the pcDNA3.1 plasmid are subjected to restriction enzyme reaction, and appropriate restriction endonucleases are selected to digest the plasmid and the target DNA fragment. Subsequently, gel electrophoresis is performed, and the target DNA fragment is cut therefrom and gel purified. The restriction enzyme sites specifically selected in the embodiment of the present invention are KpnI and XhoI.

[0059] (5) Ligation reaction:

[0060] The pcDNA3.1 plasmid that has been digested and purified is ligated with the target DNA fragment, and DNA ligase is usually used to connect the two into a complete recombinant plasmid.

[0061] 2. Conversion and screening:

[0062] The ligated recombinant plasmid is transformed into a suitable host bacterium, such as Escherichia coli (E. coli), and screened on an agar plate containing appropriate antibiotics to obtain a positive clone containing the target insert.

[0063] 3. Sequencing verification:

[0064] The selected positive clones were sequenced to confirm that the target gene sequence had been correctly inserted into the pcDNA3.1 plasmid and had no mutations. The primers for sequencing verification are as follows:

[0065] PRIMER1:22bp:5'–CTAATACGACTCACTATAGGGC-3'(SEQ ID NO.7);

[0066] PRIMER2: 23bp: 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO. 8).

[0067] Large-scale culture and extraction: The verified positive clones are cultured in large quantities, and a suitable method (such as the bovine heparin method) is used to extract a large amount of pure pcDNA3.1-target gene recombinant plasmid.

[0068] The above steps are general operation procedures based on pcDNA3.1 plasmid construction. The specific operations can be adjusted and optimized according to experimental requirements and specific gene sequences.

[0069] 4. Cell transfection:

[0070] The steps for cell transfection based on pcDNA3.1 are usually as follows:

[0071] (1) Prepare transfection complexes:

[0072] Mix pcDNA3.1 plasmid DNA with a transfection reagent (such as lipofectamine) according to the manufacturer's recommended ratio and incubate in an appropriate buffer to form a transfection complex.

[0073] (2) Treatment of target cells:

[0074] The target cells to be transfected (typically cells in culture) are treated, such as by washing them and suspending them in an appropriate cell culture medium.

[0075] (3) Transfection treatment:

[0076] Add the transfection complexes formed in step 1 to the treated target cells. Usually, gently shake the culture dish after adding the complexes to ensure that they are fully and evenly distributed.

[0077] (4) Incubate cells:

[0078] Incubate the treated cells in a 37°C incubator for a certain period of time, usually 4-6 hours, to promote DNA internalization and expression.

[0079] (5) Replacement of culture medium:

[0080] After the incubation period, consider replacing the medium with antibiotics to select and maintain cells expressing pcDNA3.1.

[0081] (6) Collection and analysis:

[0082] Depending on the experimental design, cells can be collected at an appropriate time point after transfection for RNA or protein analysis to verify the expression effect and function of pcDNA3.1.

[0083] Example 3: Single-cell sequencing

[0084] The experimental steps for single-cell transcriptome sequencing on the 10X platform in the present invention generally include the following main stages:

[0085] 1. Cell sample preparation:

[0086] Collect and process cell samples to ensure the health of cells and the integrity of single cells. Usually, cell lysis and centrifugation are required to obtain single-cell suspension.

[0087] 2. Cell sorting and fixation:

[0088] Using cell sorting techniques (such as flow cytometry or microfluidics), single cells are sorted into tiny reaction containers (such as microfluidic chips or 96-well plates), where each single cell is fixed to maintain the integrity of its RNA.

[0089] 3. Cell lysis and RNA extraction:

[0090] The fixed single cells are lysed to release RNA, and RNA is extracted using an RNA extraction kit. This step requires ensuring the high quality and integrity of the RNA to ensure the accuracy of subsequent transcriptome analysis.

[0091] 4. Reverse transcription and cDNA synthesis:

[0092] Reverse transcriptase is used to transcribe RNA extracted from single cells into cDNA. During this process, a specific barcode sequence may be added to label the RNA of each single cell.

[0093] 5. Library construction:

[0094] Library construction of synthesized cDNA usually involves steps such as fragmentation, end repair, linker ligation, and PCR amplification to prepare the library for sequencing.

[0095] 6. Sequencing platform:

[0096] The constructed single-cell transcriptome library is loaded onto the 10X platform for high-throughput sequencing. During the sequencing process, the cDNA of each single cell will be randomly assigned to different sequencing reads while retaining the Barcode information.

[0097] 7. Data analysis:

[0098] The sequencing data are subjected to bioinformatics analysis such as quality control, alignment, single-cell expression profile analysis and clustering, which will reveal the gene expression pattern of each single cell and its location and function in the cell population.

[0099] 8. Results interpretation and verification:

[0100] Interpret the cell type, state, and role in biological processes based on the analysis results. Validation experiments, such as single-cell PCR or immunohistochemistry, may be required to confirm the accuracy and reproducibility of the analysis results. These steps constitute the basic experimental process of 10X platform single-cell transcriptome sequencing, which can deeply reveal the gene expression characteristics of individual cells and provide important data support for understanding cell types and functions.

[0101] 9. Data Analysis:

[0102] The specific data analysis steps of transcriptome expression sequence tags in cell lineage tracing usually involve the following key aspects:

[0103] (1) Data quality control and preprocessing: First, the raw data obtained from the sequencing platform is quality assessed and quality controlled. This includes checking sequence quality, removing low-quality sequences and adapter sequences, and removing PCR duplicate sequences to ensure the reliability and accuracy of subsequent analysis.

[0104] (2) Sequence alignment and quantification:

[0105] Align the cleaned sequence to the reference genome or transcriptome using professional alignment tools such as Bowtie, STAR, etc. After alignment, calculate the expression of each gene based on the alignment results, usually using FPKM (number of expected fragments per million base pairs) or TPM (number of transcripts per million transcriptomes) as units for quantification of expression levels.

[0106] (3) Analysis of differentially expressed genes:

[0107] Statistical methods (such as DESeq2, edgeR, etc.) are used to compare samples under different conditions and identify differentially expressed genes. These genes may show significant expression differences in different cell lineages or states, which helps to understand lineage-specific gene expression patterns.

[0108] (4) Cell lineage analysis and clustering:

[0109] Based on the data of differentially expressed genes, cell clustering analysis can be performed. Clustering analysis can group cells with similar expression patterns into different cell populations or lineages. This step can be achieved through dimensionality reduction techniques such as principal component analysis (PCA) or popular nonlinear dimensionality reduction methods such as t-SNE or UMAP.

[0110] (5) Marker gene identification:

[0111] After different cell populations or lineages have been identified, marker genes specific to each population or lineage can be identified and validated. Marker genes are genes that are highly expressed in a specific cell type or state and are often used to identify and distinguish different cell populations.

[0112] (6) Functional enrichment analysis:

[0113] Functional enrichment analysis of differentially expressed genes can reveal biological functions and pathways that are significantly enriched in specific cell lineages, which helps to understand the functional characteristics of different lineages and their roles in tissue or organ development.

[0114] (7) Network analysis and data visualization:

[0115] Finally, the analysis results are presented using bioinformatics tools and data visualization techniques, such as gene co-expression network analysis, pathway maps, or heat maps. These visualization tools help to intuitively display the relationships and functional differences between different cell lineages.

[0116] The results of single-cell transcriptome sequencing analysis based on clustering and expression profile tags are as follows: Figure 5 As shown, after adding expression profile tags, specific single cells can be identified through random sequence barcodes, and changes in cells from the same lineage and changes in target genes can be observed at different time points.

[0117] In summary, data analysis of transcriptome expression sequence tags in cell lineage tracing is a complex and systematic process that requires the combination of multiple bioinformatics techniques and tools to deeply understand the expression characteristics and functional roles of cells under different conditions.

[0118] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0119] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An oligonucleotide for labeling cells, characterized in that The nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.1, and the oligonucleotide comprises, from the 5' end to the 3' end: Primer 1, barcode, primer 2, Poly A tail and tailing signal, the nucleotide sequences of primer 1 and primer 2 are shown in SEQ ID NO.2-SEQ ID NO.3 respectively, and the barcode consists of 10 random bases.

2. An expression vector for labeling cells, characterized in that: The expression vector contains the oligonucleotide for labeling cells according to claim 1.

3. A method for constructing an expression vector for labeling cells according to claim 2, characterized in that: The method comprises: Synthetically obtaining the oligonucleotide for labeling cells as claimed in claim 1; amplifying the oligonucleotide by PCR to obtain a PCR product; The PCR product is connected to an expression vector to obtain an expression vector for labeling cells.

4. A method for labeling cells, characterized in that: The method comprises: The expression vector for labeling cells according to claim 2 is transfected into cells cultured in vitro.

5. A method for constructing a high-throughput single-cell full-length transcriptome sequencing library, characterized in that: The method comprises: First, labeling cells using the method of claim 4 to obtain labeled single cells; The labeled single cells are sequentially lysed to extract RNA, the RNA is transcribed into cDNA using a reverse transcriptase, and a library is constructed for the cDNA to obtain a single-cell transcriptome sequencing library that can be directly used for subsequent sequencing.

6. A high-throughput single-cell full-length transcriptome sequencing library prepared by the method of claim 5.

7. Use of the high-throughput single-cell full-length transcriptome sequencing library according to claim 6 in single-cell sequencing.

8. A high-throughput single-cell sequencing method, characterized in that: The method comprises using the high-throughput single-cell full-length transcriptome sequencing library described in claim 6, and then using a high-throughput sequencing platform for sequencing.

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