A lineage tracing method to detect changes in the distribution of histone modifications in cells

Abstract

This invention discloses a lineage tracing method for detecting changes in the distribution of histone modifications in cells. The method can clarify the lineage relationship between cells at two time points by tracking barcodes, accurately track the phenotypic information of a cell at different time points, and achieve time-axis analysis. This method can simultaneously track changes in the distribution of multiple histone modifications in cells during embryonic development, cell differentiation, or tumor evolution.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a lineage tracing method for detecting changes in the distribution of histone modifications in cells. Background Technology

[0002] The application of single-cell technology provides a broad platform for studying embryonic development and tumors. Single-cell ATAC-seq (chromatin accessibility sequencing) and single-cell CUT&Tag (targeted splicing and transposition technology) have provided powerful support for studying chromatin state, histone modifications, and their roles in embryonic development, tumor progression, and drug resistance at the single-cell level. SHARE-seq (simultaneous high-throughput ATAC and RNA expression with sequencing) measures chromatin accessibility and gene expression in the same single cell, revealing that chromatin accessibility precedes gene expression during lineage determination, thus preparing the cell lineage for identification. Jindal et al. developed CellTag-multiulti single-cell lineage tracking technology, which can measure chromatin state and RNA transcriptome of the same cell and track the dynamic changes of these cells during development. By combining CellTag-multi with scRNA-seq (single-cell transcriptome sequencing) and scATAC-seq (single-cell chromatin accessibility sequencing), they achieved independent clone tracking of transcription and chromatin open / closed states, and successfully used it to characterize the progenitor cell lineage initiation process in mouse hematopoiesis. In 2021, Gopalan et al. developed Multi-CUT&Tag (multi-antibody-mediated targeted cleavage and transposition) technology by equipping different antibodies with different adapter barcodes, achieving a breakthrough in detecting two histone modifications in a single cell. Recently, the newly developed uCoTarget (ultra-high-throughput co-labeling and enrichment of multiple epigenetic proteins in the same cell) technology has enabled ultra-high-throughput single-cell co-analysis of multiple epigenetic modification proteins at the single-cell level. However, none of the existing methods can track changes in the distribution of cellular histone modifications on DNA during embryonic development or tumor evolution. There is currently no method to monitor the dynamic changes in the chromatin localization of histone modifications in cells during cell differentiation or tumor evolution. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method suitable for simultaneously tracking multiple histone modification changes in cells at the single-cell level. This method can simultaneously track changes in the distribution of multiple histone modifications during embryonic development or tumor evolution. To achieve the above objective, the present invention employs the following technical solution:

[0004] On one hand, the present invention provides a lineage tracing method for detecting changes in the distribution of histone modifications in cells, comprising:

[0005] S1. Label the cells to be tested with lineage tracking barcodes;

[0006] S2. After the test cells marked with lineage tracking barcodes in S1 are expanded by one generation, they are divided into two parts. One part is recorded as the T0 time point cells, and the other part is differentiated or treated. Then, the cells are collected at the T1 time point where they need to be tested and recorded as the T1 time point cells.

[0007] S3. Perform single-cell CUT & Tag sequencing on cells at time points T0 and T1;

[0008] S4. Based on the single-cell CUT & Tag sequencing results in S3, analyze the phylogenetic relationship and epigenetic characteristics of cells at time points T0 and T1;

[0009] The lineage tracing barcode and the antibody-specific barcode used in the single-cell CUT&Tag sequencing are respectively linked to the sequencing primer binding portions read1 and read2, which are completely identical in nucleic acid sequence.

[0010] In the method of the present invention, the lineage tracing barcode and the antibody-specific barcode used in the single-cell CUT&Tag sequencing can be captured simultaneously in the single-cell CUT&Tag sequencing.

[0011] In some implementations, the phylogenetic barcode consists of 6 units, each containing 3 random bases and 2 fixed bases, with the fixed bases maintaining an ATCG balance. When the required tracking barcode is large, it can be increased to a 7-unit barcode (each containing 4 random bases and 2 fixed bases).

[0012] In some embodiments, the nucleotide sequence of read1 is shown in SEQ ID NO.1, and the nucleotide sequence of read2 is shown in SEQ ID NO.2.

[0013] In some implementations, step S1 includes inserting a gene construct for cell labeling to trace cell lineages into the genome of the cell to be tested, said gene construct consisting of a lineage tracking barcode and sequencing primer binding portions read1 and read2.

[0014] In some implementations, the gene construct includes a promoter, a reporter gene, read1, a lineage tracking barcode, read2, a reverse transcription primer binding site, and a polyadenylate tail (polyA).

[0015] In some implementations, the reverse transcription primer binding site can be replaced with a primer sequence that does not bind to the genome of a human or other research species. This primer sequence, which does not bind to the genome of a human or other research species, can be used in a 10x system to add a 10x barcode to the multiCUT&Tag product and linearly amplify it while simultaneously exponentially amplifying the tracking barcode, thereby increasing the probability of detecting the tracking barcode.

[0016] In some implementations, the reporter gene is a fluorescent protein gene.

[0017] In this invention, the structure of the gene construct is as follows: Figure 1 As shown in B.

[0018] In some implementations, step S1 includes transfecting host cells with an expression vector and a lentiviral packaging vector loaded with the said gene construct to obtain lentiviral particles containing the said gene construct, and then infecting the cells to be tested with the lentiviral particles.

[0019] In some implementations, when infecting the test cells with the lentiviral particles, the infection rate is controlled to be below 30% to reduce the probability of two viruses infecting the same cell.

[0020] In some implementations, when transfecting host cells with the expression vector and lentiviral packaging vector loaded with the aforementioned gene construct, the transfection ratio of host cells to expression vector is 1 × 102. 7 Each host cell corresponds to 20 μg of expression vector.

[0021] In some implementations, the method for constructing the expression vector includes ligating the gene construct into the vector via enzyme digestion.

[0022] In some embodiments, step S1 further includes amplifying the expression vector before transfecting host cells. In a preferred embodiment, the method for amplifying the expression vector includes conversion into *E. coli* via electroporation for amplification.

[0023] In some embodiments, the method for amplifying the expression vector further includes spreading the transformed *E. coli* on the surface of a solid culture medium, with a colony density of 90-110 colonies per square centimeter. As a preferred embodiment, it is necessary to ensure that the total colony count matches the richness of the lineage tracking barcode; for example, 7 units (each unit containing 4 random bases + 2 fixed bases) require 0.5-1 × 10⁻⁶ colonies. 6 One colony.

[0024] In some implementations, after culturing for 1 day at 30°C or room temperature, liquid culture medium is added to the surface of the solid culture medium, the colonies are scraped into the liquid culture medium, and the culture is carried out in a shaker at 37°C until the OD value is about 0.6, and the bacterial cells are collected to extract plasmids.

[0025] In some implementations, in step S2, the test cells marked with lineage tracking barcodes in S1 are amplified one generation and then divided into two portions.

[0026] In some implementations, the single-cell CUT&Tag sequencing is multi-antibody-mediated single-cell CUT&Tag sequencing.

[0027] In some implementations, the single-cell CUT&Tag sequencing uses a multi-antibody-guided Tn5 digestion of an antibody-linker containing antibody-specific barcodes targeting the target protein-pAG (proteins A and G)-Tn5 (transposase 5) complex.

[0028] In a specific embodiment of the present invention, the antibody-adaptor containing antibody-specific barcode-pAG-Tn5 complex targeting the target protein comprises an antibody targeting the target protein, a pAG-Tn5 transposase, and an adaptor containing antibody-specific barcode. The adaptor containing antibody-specific barcode is a DNA strand, comprising a sequencing primer binding portion (read1 or read2), an antibody-specific barcode, and an ME sequence, wherein the ME sequence portion is double-stranded DNA, and the remainder is single-stranded. The structure of the antibody-adaptor containing antibody-specific barcode-pAG-Tn5 complex of the present invention is as follows: Figure 2 As shown.

[0029] In a specific embodiment of the present invention, the antibody-linker containing antibody-specific barcode-pAG (proteins A and G)-Tn5 (transposase 5) complex targeting the target protein is prepared by the following method:

[0030] (1) A 2×strep tag was fused to the N-terminus of pAG-Tn5 transposase to construct a recombinant expression vector and transform it into an E. coli expression system for induction and purification to obtain 2×strep-pAG-Tn5 protein;

[0031] (2) Each antibody targeting the target protein is equipped with a adapter combination containing antibody-specific barcodes. The adapter combination includes adapter A and adapter B. Adapter A contains a single strand of DNA consisting of a sequencing primer binding part read1, a first antibody barcode sequence, and a ME-rev complementary sequence. Adapter B contains a single strand of DNA consisting of a sequencing primer binding part read2, a second antibody barcode sequence, and a ME-rev complementary sequence. Adapter A and adapter B are annealed with complementary ME-rev sequences and then loaded onto the 2×strep-pAG-Tn5 protein prepared in step (1) to obtain a 2×strep-pAG-Tn5 protein that has been bound to adapter A and adapter B.

[0032] (3) According to the molar ratio of 2×strep-pAG-Tn5 protein to antibody targeting the target protein = 2:1, the antibody targeting the target protein is loaded onto the 2×strep-pAG-Tn5 protein that has bound adapter A and adapter B to form an antibody targeting the target protein-adaptor containing antibody-specific barcode-pAG (protein A and G)-Tn5 (transposase 5) complex.

[0033] In this invention, the nucleotide sequence of ME-rev is shown in SEQ ID NO.19.

[0034] In some implementations, the single-cell CUT&Tag technology includes the following steps: centrifuging cells to remove supernatant, washing with a first buffer (antibody buffer), adding the first buffer and an antibody targeting the target protein—a linker containing an antibody-specific barcode—to the cell nucleus for incubation, centrifuging again to remove supernatant, washing with a second buffer, and then adding Mg... 2+ The cells were incubated with a second buffer to activate the digestion activity of the Tn5 transposase, and then a neutralization buffer was added to terminate the digestion reaction. After the digestion reaction was terminated, the cells were used to construct a library and sequence the cells.

[0035] As a preferred embodiment, the first buffer contains 20 mM HEPES·NaOH (pH 7.2-7.4), 150 mM NaCl, 0.5 mM spermidine, 1 mM EDTA, 0.05% (w / w) digitalis saponins, 0.01% (v / v) NP40, 1%-2% (w / w) BSA, and 1× protease inhibitor.

[0036] As a preferred embodiment, the second buffer solution comprises 20 mM HEPES·NaOH (pH 7.2-7.4), 300 mM NaCl, 0.5 mM spermidine, 1 mM EDTA, 0.05% (w / w) digitalis saponins, 0.01% (v / v) NP40, 1%-2% (w / w) BSA, and 1× protease inhibitor.

[0037] As a preferred embodiment, 10 mM MgCl2 is added to the second buffer solution to obtain the Mg-added solution. 2+ In the second buffer solution.

[0038] As a preferred embodiment, the neutralization buffer is prepared by adding 10 μL of 0.5M EDTA to every 200 μL of the second buffer.

[0039] In some implementations, step S4 includes: identifying the cell to which the DNA fragment belongs based on the cell barcode; determining the epigenetic modification information of histones binding to the DNA fragment through antibody-specific barcodes; and clarifying the cell identity by combining the lineage tracing barcode and reconstructing the lineage relationship between cells at time points T0 and T1.

[0040] In some implementations, during data analysis, algorithms can be used to remedy the situation where two lentiviruses simultaneously infect a single cell: the tracking barcodes carried by each cell are enumerated based on the cell's 10× barcode, and tracking barcodes with fewer than 2 copies obtained from sequencing are discarded. Cells carrying more than 2 identical barcodes can be recorded as originating from the same cell, thereby optimizing the calculation results for a single cell infecting multiple tracking barcodes.

[0041] In another aspect, the present invention provides a kit comprising the gene construct described above and the antibody-linker containing antibody-specific barcode-pAG (proteins A and G)-Tn5 (transposase 5) complex targeting the target protein described above.

[0042] In some implementations, the kit further includes the first buffer, the second buffer, and the neutralization buffer described above.

[0043] The present invention also provides for the application of the kit in any of the following:

[0044] (1) Prepare products that simultaneously track changes in the distribution of multiple histone modifications in cells during embryonic development or tumor evolution;

[0045] (2) Detect lineage to track changes in histone modification distribution in cells.

[0046] In this invention, the product includes a reagent kit.

[0047] Beneficial effects:

[0048] This invention designs a lineage tracing system for simultaneous detection of multiple histone modifications: ① The design of the antibody-specific barcode adapter ensures that it can be simultaneously adapted to the 10×GEM single-cell ATAC system and efficiently recognized by the Tn5 enzyme; ② The DNA structure design at both ends of the tracing barcode matches the structure of the final library formed by the antibody-specific barcode adapter, so as to meet the requirements of simultaneous amplification and sequencing of cell tracing barcodes and single-cell CUT&Tag products.

[0049] This invention applies lineage tracing combined with multi-antibody-mediated single-cell CUT&Tag technology to analyze changes in histone modifications over time and with differentiation stimulation during embryonic development, and to analyze the role of histone modifications in tumorigenesis, tumor drug resistance, and tumor evolution.

[0050] Current systems for tracking cell epigenetics can only track the open or closed state of chromatin, and cannot provide information on the distribution of different histone modifications on the genome. This invention can use a small number of cells to track the distribution of each histone on the DNA.

[0051] Current techniques for simultaneously detecting the distribution of multiple histone modifications on DNA in cells can only perform many-to-many data analysis on information from cells detected at different time points. However, this invention can clarify the lineage relationship between cells at two time points by tracking barcodes, accurately track the phenotypic information of a cell at different time points, and achieve analysis along the timeline.

[0052] This invention utilizes lineage tracing to accurately detect the determining role of histone modifications in tumor cell metastasis phenotype during breast cancer cell metastasis. Attached Figure Description

[0053] Figure 1 The adapter structure, library structure, and plasmid library structure of this application are shown, wherein, Figure 1 Figure A illustrates the adapter design carrying antibody-specific barcodes and the library structure generated by single-cell CUT&Tag mediated by single-cell multi-antibody synthesis. Figure 1 Figure B illustrates a plasmid library design carrying a tracking barcode. Figure 1 C shows the library structure for tracking barcodes in single-cell sequencing;

[0054] Figure 2 The structure of the antibody-linker containing antibody barcode-pAG-Tn5 complex is shown;

[0055] Figure 3This demonstrates a data analysis strategy that distinguishes cells based on 10× barcodes and then distinguishes histone modification information on DNA fragments based on antibody-specific barcodes.

[0056] Figure 4 This paper demonstrates a data analysis strategy that distinguishes cells based on 10× barcodes, identifies cells based on tracking barcodes, and reconstructs the phylogenetic relationships between cells at different time points based on cell identification information.

[0057] Figure 5 This illustrates the experimental design and results of detecting changes in the epigenetic modification states of multiple histones during breast cancer cell metastasis in Example 2. Figure 5 Figure A illustrates the experimental design for detecting changes in the epigenetic modification states of multiple histones during breast cancer cell metastasis. Figure 5 Figure B shows the clonal distribution of tumor cells in the in situ tumor and metastatic tissues of the lung, liver, and lymph nodes. Figure 5 The image shows the distribution characteristics of H3K27me3 in breast cancer cells with metastatic potential, and the overlap of differential peaks of H3K27me3 in lung metastases, liver metastases, and lymph node metastases. Figure 5 The diagram in Figure D shows the genomic distribution of H3K27me3 features in breast cancer cells with metastatic potential. Figure 5 The image shows the distribution characteristics of H3K27ac in breast cancer cells with metastatic potential, and the overlap of H3K27ac differential peaks in lung metastases, liver metastases, and lymph node metastases. Figure 5 The middle F shows the genomic distribution of H3K27ac features in breast cancer cells with metastatic potential;

[0058] Figure 6 The quality control results of plasmids obtained by enzyme digestion and ligation are shown.

[0059] Figure 7 The effect of BSA addition on the nuclear state of cells after Tn5 digestion is shown. Figure 7 Image A shows the morphology of cell nuclei after enzyme digestion in standard CUT & Tag buffer. Figure 7 Figure B shows the morphology of cell nuclei after enzyme digestion in CUT&Tag buffer containing BSA. Detailed Implementation

[0060] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0061] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0062] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0063] To monitor the dynamic changes in histone modifications during cell differentiation or tumor evolution, this invention develops a novel technique that can simultaneously track the distribution changes of multiple histone modifications on DNA. A lineage-tracking lentiviral system targeting the simultaneous detection of multiple histone modifications is constructed. Each cell in the cell population to be monitored is labeled with a unique tracking barcode. After one generation of cell expansion, the cells are divided into two equal parts: one part records the epochal state at time T0, and the other part is further differentiated or treated. At time T1, cells are collected again and their epochal state is detected. Using an in vitro assembled antibody-antibody-specific barcode-pAG (proteins A and G)-Tn5 (transposase 5) complex, T0 and T1 cells are subjected to multi-antibody-guided Tn5 digestion. The cells are then labeled with 10× barcodes using a 10× Genomics single-cell ATAC system. All DNA fragments are then pooled, a library is constructed using universal primers, and next-generation sequencing is performed using self-provided primers. Sequencing results use 10× barcodes to identify the cells to which DNA fragments belong, antibody-specific barcodes determine histone epigenetic modifications on these DNA fragments, and cell tracking barcodes identify the cells and reconstruct the lineage relationships between cells at T0 and T1 time points. This achieves the goal of tracing the changes in multiple histone epigenetic modification states during embryonic development and tumor evolution.

[0064] Example 1

[0065] (1) Design and construction of a lineage tracing system for simultaneous detection of multiple histone modifications:

[0066] 1) MultiCUT&Tag adapter design: The multiCUT&Tag adapter must have an ME sequence (transposon recognition sequence) adapted to Tn5 (transposase 5) and an adapter sequence adapted to the 10×Genomics ATAC system, and also have a recognizable antibody barcode sequence (e.g., Figure 1 (As shown in A).

[0067] 2) Plasmid Library Design and Construction: The tracking barcode consists of several sets of random and fixed bases. The cell tracking barcode must be able to be simultaneously amplified and sequenced with the multiCUT&Tag product. Therefore, both ends of the barcode must have amplification and sequencing sequences identical to the multiCUT&Tag product, i.e., read1 and read2. To facilitate the screening of cells carrying the tracking barcode, the above sequence is placed at the 3' end of the reporter gene GFP expression frame. Furthermore, a reverse transcription primer binding site is added between the polyA tails of read2 and GFP mRNA. The GFP promoter, expression frame, read1, cell tracking barcode, and read2 are loaded together into a lentiviral expression vector to form a plasmid library of the tracking system (e.g., ...). Figure 1 (As shown in Figure B). Reads1 and 2 (sequencing primers), and N5 and N7 (universal library construction primers) were used sequentially for library construction PCR. The resulting library was then subjected to next-generation sequencing to assess the diversity and uniformity of tracking barcodes in the plasmid library. The structure of the tracking barcodes after library construction using 10× single-cell CUT&Tag is shown below. Figure 1 As shown in C. The sequencing adapters at both ends of the tracking barcode are shown in Table 1.

[0068] Table 1 Sequencing adapters at both ends of the tracking barcode

[0069] sequence SEQ ID NO. Read1 <![CDATA[ TCGTCGGCAGCGTC TCCACGC]]> 1 Read2 <![CDATA[ GTCTCGTGGGCTCGG CTGTCCCTGTCC]]> 2

[0070] The underlined sequences in Table 1 are those that are associated with 10xbeads.

[0071] 3) Lentiviral Packaging and Infection: HEK293T cells (human embryonic kidney cells) were transfected with an expression vector containing the GFP promoter and expression cassette -Read1-tracking barcode-Read2-polyA, and a lentiviral packaging vector at a ratio (in a three-plasmid packaging system, the expression plasmid: packaging plasmid pSPAX2 and pMD.2g = 10:7.5:2.5 (mass ratio)). Virus-containing culture supernatant was collected at 24 and 48 hours. After removing cell debris, the virus pellet was obtained by ultracentrifugation and dissolved in PBS. The MOI (Mean Intake) of research subjects such as embryonic stem cells or tumor cells was tested. A formal infection concentration of 16%-21% was selected for the experiment to ensure that each cell obtained only one tracking barcode. Then, eGFP (green fluorescent protein) positive cells were sorted by flow cytometry. After expanding the cell bank, 1 million cells were sequenced using tracking barcodes to detect the richness and uniformity of tracking barcode distribution in the cell population.

[0072] 4) Collect cells at different time points to form single-cell CUT & Tag: Take an appropriate number of cells from the cell bank based on the richness of the tracking barcodes. Before processing, culture the cells normally for one generation to divide them into two copies. After thorough digestion, take half of the cells for single-cell CUT & Tag at time point T0, and perform other operations such as induction of differentiation or drug treatment on the other half of the cells. At the T1 time point where detection is required, take an equal amount of cells to form single-cell CUT & Tag.

[0073] (2) Multi-antibody mediated single-cell CUT&Tag technology solution:

[0074] 1) Preparation of the antibody-adaptor-PAG-Tn5 complex for multi-antibody-mediated CUT&Tag: A 2×strep tag was added to the N-terminus of the original pAG-Tn5 (protein A-protein G-transposase 5 fusion protein). The plasmid was transfected into BL21(DE3) competent cells to express and purify the strep tag-PAG-Tn5 protein. Each antibody was equipped with two antibody barcode sequences, adaptor A and adaptor B (as shown in Table 2). Adaptor A and adaptor B were mixed with ME-rev at a molar ratio of 1:1 and annealed. The two annealing products were then loaded onto pAG-Tn5 at a ratio of 3:3:1. Subsequently, the corresponding antibody was loaded at a molar ratio of PAG-Tn5:antibody = 2:1. The antibody-adaptor-PAG-Tn5 complex containing antibody barcodes was incubated with streptavidin magnetic beads to bind. After washing away unbound adapters and antibodies, the antibody-adaptor-PAG-Tn5 complex was eluted with PBS containing 25 mM biotin. Biotin was then removed from the PBS by ultrafiltration. The antibody-adaptor-PAG-Tn5 complex is shown below. Figure 2 As shown.

[0075] Table 2. Sequences of adapter A and adapter B required for preparing the antibody-barcode-carrying adapter (including the antibody barcode adapter)-PAG-Tn5 complex.

[0076]

[0077]

[0078] The bolded items in Table 2 are antibody indexes.

[0079] 2) Multi-cell CUT & Tag: Starting with 250,000 cells, after centrifugation and removal of supernatant, use antibody buffer (20 mM MEPES·NaOH pH 7.4, 150 mM NaCl, 0.5 mM spermidine, 1 mM EDTA, 0.05% (w / v) digitalis saponins, 0.01% (v / v) NP40, 1% (w / v) BSA, 1× protease inhibitor (InStab)TM After washing once with a Protease Inhibitor Cocktail (yeason, 20124ES03) diluted 1:100, add 200 μL of antibody buffer and 2 μg of each antibody-barcode-carrying adapter-PAG-Tn5 complex to 250,000 cell nuclei and incubate overnight at 4°C. Centrifuge at 600g for 3 minutes, discard the supernatant, and wash the cells three times with 200μL Dig-300-BSA buffer (20mM HEPES NaOH pH 7.4, 300mM NaCl, 0.5mM spermidine, 1mM EDTA, 0.05% digitalis saponin, 0.01% NP40, 1% BSA, 1× protease inhibitor). Resuspend the cells in 200μL Dig-300-BSA buffer containing 10mM MgCl2, incubate at 37℃ for 1 hour, and then add 200μL neutralization buffer (add 10μL 0.5M EDTA per 200μL Dig-300-BSA buffer) to terminate the enzyme digestion reaction. After centrifugation at 600g for 3 minutes at room temperature, the cells were resuspended in 200 μL of 1×DNB buffer containing 1% BSA, as per the scATAC-seq kit (v2) (Chromium Next GEM Single Cell ATACReagent Kits v2). Larger particles were removed by filtering through a 40 μm cell strainer. 5 μL of cells were mixed with 5 μL of trypan blue staining solution, and the cell nuclei were observed and counted for morphology. Water-in-oil PCR and subsequent library construction were performed according to the Chromium Next GEM Single Cell ATAC Reagent Kits v2 (10× company's single-cell chromatin accessibility assay kit, version 2).

[0080] (3) Data analysis strategy:

[0081] 1) Based on the information in the I1 file obtained from sequencing (i.e., 10× barcode information), the sequencing data is distributed to each cell, and cells that do not meet the standards are removed according to the standard single-cell cut & tag process. Figure 3 ).

[0082] 2) Based on the antibody-specific barcode information on the DNA fragments obtained from sequencing, the sequenced DNA fragments of each cell are further divided into datasets for each histone modification. Figure 3 ).

[0083] 3) Extracting the tracking barcode information from the cell: Based on the DNA sequences on both sides of the tracking barcode, extract the DNA fragment containing the tracking barcode information. Figure 4Based on the 10× barcode information of I1 obtained from sequencing, the cell scCUT&Tag data and the cell tracking barcode were matched. Cells with less than 2 DNA fragments obtained from sequencing the tracking barcode were discarded and not subjected to subsequent pedigree analysis.

[0084] 4) Reconstruct the phylogenetic relationships of cells at different time points using single-cell CUT & Tag lineage tracking barcodes at T0 and T1 time points. Based on the tracking barcodes of cells with different phenotypes at T1 time point, the epigenetic characteristics of cells possessing that tracking barcode at T0 time point can be traced back. Figure 4 ).

[0085] Example 2 uses the method of the present invention to accurately detect the determining role of histone modifications on tumor cell metastasis phenotype during breast cancer cell metastasis by applying lineage tracing of changes in multiple histone epigenetic modification states.

[0086] (1) Experimental steps:

[0087] 1) Lentiviral Packaging and Infection: Expression vectors loaded with eGFP-CUT&Tag tracking barcodes and lentiviral packaging vectors were transfected into HEK293T cells (human embryonic kidney cells) at a ratio of 10:7.5:2.5 (mass ratio) using a three-plasmid packaging system. Virus-containing culture supernatants were collected at 24 and 48 hours. After removing cell debris, the virus pellet was obtained by ultracentrifugation and dissolved in PBS. The MOI of MDA-MB-231 tumor cells was tested. A 15% infection concentration was selected for the formal infection experiment to ensure that each cell acquired only one tracking barcode. Three days after infection, eGFP (green fluorescent protein) positive MDA-MB-231 cells were sorted by flow cytometry to obtain a cell bank where each cell carried a unique tracking barcode.

[0088] 2) Tumor Implantation and Tissue Collection: After limited expansion (4-6 passages) of cells carrying tracking barcodes, half of the cells (approximately 10^6 cells) were divided into 10 portions and orally implanted into the mammary glands of 10 immunodeficient mice. After 4 weeks, tumor cells metastasized. The mice were euthanized and dissected to remove the orthotopic tumors, lungs, livers, and lymph nodes for single-cell dissociation. The obtained single-cell suspension was filtered through a cell strainer, washed twice with PBS, and resuspended to 2×10^6 cells / mL. 7 After achieving a concentration of / ml, eGFP-positive cells were screened out by flow cytometry, which identified tumor cells from in situ or metastatic lesions.

[0089] 3) Multi-antibody mediated single-cell cuts & tags:

[0090] ① Preparation of antibody-adaptor-PAG-Tn5 complex: Adaptor A-1 and Adaptor B-1 were annealed, and Adaptor A-2 and Adaptor B-2 were annealed. Antibody barcode A / B adapters and pAG-Tn5 were mixed at a molar ratio of 3:3:1 and incubated at room temperature for 1 hour. Then, Adaptor A / B1-PAG-Tn5 was mixed with H3K27me3 antibody and Adaptor A / B2-PAG-Tn5 was mixed with H3K27ac antibody at a molar ratio of pAG-Tn5:antibody = 2:1. After incubation at room temperature for 1 hour, pre-washed streptavidin magnetic beads were added for incubation and binding. After washing away unbound adapters and antibodies, the antibody-adaptor-PAG-Tn5 complex was eluted with PBS containing 25 mM biotin. The biotin in the PBS was removed by solution replacement using an ultrafiltration tube. This allows you to obtain H3K27me3 antibody-pAG-Tn5 complex carrying AdaptorA / B1 antibody barcode and H3K27ac antibody-pAG-Tn5 complex carrying AdaptorA / B2 antibody barcode.

[0091] ② Single-cell CUT & Tag: 250,000 cells were sorted from each tissue. After centrifugation and removal of supernatant, the cells were washed once with antibody buffer (20mM HEPES·NaOH pH 7.4, 150mM NaCl, 0.5mM spermidine, 1mM EDTA, 0.05% digitalis saponin, 0.01% NP40, 1% BSA, 1× protease inhibitor). 200μL of antibody buffer and 2μg of each antibody-barcode-carrying adapter-PAG-Tn5 complex to be detected were added to the cell nuclei and incubated overnight at 4°C. Centrifuge at 600g for 3 minutes, discard the supernatant, and wash the cells three times with 200μL Dig-300-BSA buffer (20mM HEPES NaOH pH 7.4, 300mM NaCl, 0.5mM spermidine, 1mM EDTA, 0.05% digitalis saponin, 0.01% NP40, 1% BSA, 1× protease inhibitor). Resuspend the cells in Dig-300-BSA buffer containing 10mM MgCl2 and incubate at 37°C for 1 hour. Terminate the enzyme digestion reaction by adding 200μL neutralization buffer (10μL of 0.5M EDTA per 200μL Dig-300-BSA buffer). Centrifuge at 600g for 3 minutes at room temperature and resuspend in 200μL 1×DNB buffer containing 1% BSA from the scATAC-seq kit (v2). Filter through a 40μm cell strainer to remove larger particles. After mixing 5 μL of cells with 5 μL of trypan blue staining solution, the cell nuclei were observed and counted. Water-in-oil PCR and subsequent library construction were performed according to the Chromium Next GEM Single Cell ATAC Reagent Kits v2 (10× company's single-cell chromatin accessibility test kit, version 2).

[0092] ③ Data Analysis: Following the standard single-cell CUT&Tag procedure, cells that did not meet the standards were removed. DNA fragments containing tracking barcode information were extracted based on the DNA sequences flanking the tracking barcode. The 10× barcode information of I1 obtained from sequencing was matched with the cell scCUT&Tag data and the cell's tracking barcode. Cells with a tracking barcode read count greater than 2 were subjected to lineage analysis. Based on the antibody-specific barcode information on the sequenced DNA fragments, the sequenced DNA fragments of each cell were further divided into datasets for each histone modification. The lineage relationship between cells in situ and metastatic lesions was reconstructed based on the tumor cell lineage tracking barcodes. Differences in the epigenomic distribution of H3K27me3 and H3K27ac between in situ cells with and without metastatic potential were analyzed.

[0093] (2) Experimental results:

[0094] Figure 5 The experimental design and results for detecting changes in the epigenetic modification status of multiple histones during breast cancer cell metastasis are presented.

[0095] ① Lineage tracking data shows that about one-third of MDA-MB-231 cells in in situ tumors will metastasize to distant sites. The most common route of metastasis to the lungs is through hematogenous spread. Clones that metastasize to the lungs and liver show similarities, but differ significantly from those that metastasize to the lymph nodes. Figure 5 (B)

[0096] ② Significant differences exist in the epigenomic distribution of H3K27me3 and H3K27ac between breast cancer cells with and without metastatic potential. The epigenomic characteristics of in situ tumor cells leading to lung, liver, and lymph node metastases share similarities and differences, with the differential distribution of H3K27me3 being more pronounced while the differences in H3K27ac are relatively smaller. The differentially distributed H3K27me3 is mainly located in intergenic regions, while the differentially distributed H3K27ac is mainly located in intronic regions.

[0097] The sequencing results using the method of this invention show that some sequencing fragments have barcodes for two different histones, which suggests that the distribution of these two histones on DNA is very similar. Therefore, in addition to simultaneously tracking the distribution changes of multiple histones, this application can also clearly identify the positional relationship of two DNA-binding proteins that are simultaneously bound at a specific DNA site in a cell, that is, to show the interaction relationship between two proteins and DNA at the single-cell level.

[0098] Comparative Example 1

[0099] During the construction of tracking barcode vectors, read1 and read2, along with the tracking barcode they carry, must be ligated into the desired vector using enzyme digestion and ligation. Homologous end ligation cannot be used to construct the vector; otherwise, the diversity of tracking barcodes will be severely compromised.

[0100] Results comparison: Vectors constructed using the homologous end ligation method were found to lack the required ligation pattern during sequencing, while clones obtained using the enzyme digestion ligation method showed a diversity of 2 × 10⁻⁶. 5 The above-mentioned diversity, and the relatively uniform distribution of TCGA bases in the barcode. The quality control results of the plasmids obtained by the enzyme digestion and ligation method are as follows: Figure 6 As shown.

[0101] Comparative Example 2

[0102] When constructing the barcode library, it is crucial to ensure that the number of barcode libraries far exceeds the number of cells to be infected, thereby minimizing the probability of two cells being infected with the same tracking barcode. The plasmid vector containing the tracking barcode needs to be transformed into *E. coli* via electroporation for amplification to ensure high transformation efficiency. The transformed bacteria should be evenly spread on 20 15cm diameter solid LB agar plates and cultured, maintaining a colony density of approximately 100 colonies per square centimeter. To avoid uneven distribution of the tracking barcode due to some colonies growing too quickly, the plates are incubated at 30°C instead of 37°C, thus maintaining the plasmid copy number at 20 × 3.14 × 7.5. 2 ×100=3.5×10 5 (This method is used in both Embodiments 1 and 2 of this application). To ensure the copy number of the tracking barcode carried by the lentiviral library, plasmid transfection and lentiviral packaging also need to be performed in a large number of 293T cells at 1×10⁻⁶ cells. 7 Transfecting cells with 20 μg of the vector plasmid and an appropriate ratio of packaging plasmids (in a three-plasmid packaging system, expression plasmid: packaging plasmids pSPAX2 and pMD.2g each = 10:7.5:2.5) yields suitable packaging efficiency. Due to the diversity of tracking barcodes at 3.5 × 10⁻⁶... 5 When infecting cells with lentiviruses, the number of successfully infected cells should be controlled to be between 1 and 2 × 10⁻⁶. 5 To reduce the proportion of two different cell lines infected with the same tracking barcode, the infection rate of cells must be kept below 30% to decrease the probability of two viruses infecting the same cell. Once the cell line carrying the tracking barcode is successfully constructed, experiments should begin as soon as possible to avoid multiple passages that could lead to an imbalance in the distribution of the tracking barcode library.

[0103] Result comparison:

[0104] When the infection rate is 30%, 70% of cells contain multiple barcodes; when the infection rate is 16%, 50% of cells contain multiple barcodes.

[0105] It is very difficult to distinguish between two different cells infected with the same tracking barcode through experimental methods, which can cause a great deviation in the experimental results.

[0106] Comparative Example 3

[0107] In the single-cell CUT&Tag experiment, the Tn5 digestion step was performed at 37°C using Dig-300-BSA buffer containing 1% BSA and 10mM MgCl2 (20mM HEPES NaOH pH 7.4, 300mM NaCl, 0.5mM spermidine, 1mM EDTA, 0.05% digitalis saponin, 0.01% NP40, 1% BSA, 1× protease inhibitor). Without BSA, cell clumping would occur, causing the single-cell CUT&Tag to fail quality control before processing and thus be unusable.

[0108] Results Comparison: Results are as follows Figure 7 As shown, Figure 7 Image A shows the state of cell nuclei after digestion with Tn5 without the addition of BSA. The cells are largely clump together, with very few single, uniform nuclei. Figure 7 The image in section B shows the state of cell nuclei after digestion with Tn5 containing BSA. The nuclei are rarely clustered and are plump.

[0109] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A lineage tracing method for detecting changes in the distribution of histone modifications in cells, comprising: S1. Label the cells to be tested with lineage tracking barcodes; S2. The test cells marked with lineage tracking barcodes in S1 are expanded and divided into two parts. One part is recorded as T0 time point cells, and the other part is differentiated or treated. Then, the cells are collected at the T1 time point where they need to be tested and recorded as T1 time point cells. S3. Perform single-cell CUT & Tag sequencing on cells at time points T0 and T1; S4. Based on the single-cell CUT & Tag sequencing results in S3, analyze the phylogenetic relationship and epigenetic characteristics of cells at time points T0 and T1; The lineage tracing barcode and the antibody-specific barcode used in the single-cell CUT&Tag sequencing are respectively linked to the sequencing primer binding portions read1 and read2, which are completely identical in nucleic acid sequence.

2. The method according to claim 1, wherein, Step S1 includes inserting a gene construct for cell labeling to trace cell lineages into the genome of the cell to be tested, wherein the gene construct consists of a lineage tracking barcode and sequencing primer binding portions read1 and read2; Preferably, the gene construct comprises a promoter, a reporter gene, read1, a lineage tracing barcode, read2, a reverse transcription primer binding site, and a polyadenylate tail (polyA). Preferably, the reporter gene is a fluorescent protein gene; Preferably, step S1 includes transfecting host cells with an expression vector loaded with the gene construct and a lentiviral packaging vector to obtain lentiviral particles containing the gene construct, and then infecting the cells to be tested with the lentiviral particles.

3. The method according to claim 2, wherein, When transfecting host cells with the expression vector and lentiviral packaging vector loaded with the aforementioned gene construct, the transfection ratio of host cells to expression vector is 1 × 10⁻⁶. 7 Each host cell corresponds to 20 μg of expression vector.

4. The method according to claim 2, wherein, The method for constructing the expression vector includes ligating the gene construct into the vector via enzyme digestion and ligation. Preferably, step S1 further includes amplifying the expression vector before transfecting the host cells. Preferably, the method for amplifying the expression vector includes converting it into Escherichia coli by electroporation for amplification; Preferably, the method for amplifying the expression vector further includes spreading the transformed Escherichia coli on the surface of a solid culture medium, with a colony density of 90-110 colonies per square centimeter. Preferably, after culturing at 30°C or room temperature for 1 day, liquid culture medium is added to the surface of the solid culture medium, the colonies are scraped into the liquid culture medium, and cultured in a shaker at 37°C until the OD value is about 0.6, and the bacterial cells are collected to extract plasmids.

5. The method according to claim 1, wherein, In step S2, the test cells marked with lineage tracking barcodes in S1 are expanded by one generation and then divided into two groups.

6. The method according to claim 1, wherein, The single-cell CUT&Tag sequencing described herein is multi-antibody-mediated single-cell CUT&Tag sequencing. Preferably, the single-cell CUT&Tag sequencing uses an antibody targeting the target protein, an adapter containing an antibody-specific barcode, and a pAG-Tn5 complex for multi-antibody-guided Tn5 digestion. Preferably, the antibody-adaptor with antibody-specific barcode-PAG-Tn5 complex targeting the target protein comprises an antibody targeting the target protein, pAG-Tn5 transposase, and an adaptor with antibody-specific barcode; the adaptor with antibody-specific barcode is a DNA strand, and the adaptor with antibody-specific barcode comprises a sequencing primer binding portion read1 or read2, an antibody-specific barcode, and an ME sequence, wherein the ME sequence portion is double-stranded DNA, and the remainder is single-stranded; Preferably, the antibody-linker containing antibody-specific barcode-pAG (proteins A and G)-Tn5 (transposase 5) complex targeting the target protein is prepared by the following method: (1) A 2×strep tag was fused to the N-terminus of pAG-Tn5 transposase to construct a recombinant expression vector and transformed into an E. coli expression system for induction expression and purification to obtain 2×strep-pAG-Tn5 protein; (2) Each antibody targeting the target protein is equipped with a adapter combination containing antibody-specific barcodes. The adapter combination includes adapter A and adapter B. Adapter A contains a single strand of DNA consisting of a sequencing primer binding part read1, a first antibody barcode sequence, and a ME-rev complementary sequence. Adapter B contains a single strand of DNA consisting of a sequencing primer binding part read2, a second antibody barcode sequence, and a ME-rev complementary sequence. Adapter A and adapter B are annealed with complementary ME-rev sequences and then loaded onto the 2×strep-pAG-Tn5 protein prepared in step (1) to obtain a 2×strep-pAG-Tn5 protein that has been bound to adapter A and adapter B. (3) According to the molar ratio of 2×strep-pAG-Tn5 protein to antibody targeting the target protein = 2:1, the antibody targeting the target protein is loaded onto the 2×strep-pAG-Tn5 protein that has bound adapter A and adapter B to form an antibody targeting the target protein-adaptor containing antibody-specific barcode-pAG (protein A and G)-Tn5 (transposase 5) complex; Preferably, the single-cell CUT&Tag technology includes the following steps: centrifuging the cells to remove the supernatant, washing with a first buffer, adding the first buffer and an antibody targeting the target protein-adaptor containing an antibody-specific barcode-pAG-Tn5 complex to the cell nucleus for incubation, centrifuging again to remove the supernatant, washing with a second buffer, adding a second buffer supplemented with Mg2+ for incubation to activate the Tn5 transposase digestion activity, and then adding a neutralization buffer to terminate the digestion reaction; constructing a library and sequencing the cells after terminating the digestion reaction. Preferably, the first buffer solution contains 20 mM HEPES·NaOH (pH 7.2-7.4), 150 mM NaCl, 0.5 mM spermidine, 1 mM EDTA, 0.05% digitalis saponins, 0.01% NP40, 1%-2% BSA, and 1× protease inhibitor. Preferably, the second buffer solution contains 20 mM HEPES·NaOH (pH 7.2-7.4), 300 mM NaCl, 0.5 mM spermidine, 1 mM EDTA, 0.05% digitalis saponin, 0.01% NP40, 1%-2% BSA, and 1× protease inhibitor. Preferably, 10 mM MgCl2 is added to the second buffer solution to obtain the second buffer solution with added Mg2+. Preferably, the neutralization buffer is prepared by adding 10 μL of 0.5M EDTA to every 200 μL of the second buffer.

7. The method according to any one of claims 1-6, wherein, Step S4 includes: identifying the cell to which the DNA fragment belongs based on the cell barcode; determining the epigenetic modification information of histones on the DNA fragment by using antibody-specific barcodes; clarifying the cell identity by combining the lineage tracing barcode and reconstructing the lineage relationship between cells at time points T0 and T1.

8. A kit comprising the gene construct of claim 2 and the antibody-adaptor containing antibody-specific barcode-pAG-Tn5 complex targeting a target protein of claim 6.

9. The kit according to claim 8, further comprising the first buffer, second buffer, and neutralization buffer as described in claim 6.

10. The use of the kit according to claim 8 or 9 in any of the following: (1) Prepare products that simultaneously track changes in the distribution of multiple histone modifications in cells during embryonic development or tumor evolution; (2) Detect lineage to track changes in histone modification distribution in cells.

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