A method for obtaining human bone tissue cells suitable for whole transcriptome sequencing and its application

Abstract

The invention discloses a method for obtaining human sclerosing bone tissue cells suitable for full transcriptome sequencing, comprising the following steps: tissue cleaning and pretreatment: placing human sclerosing bone tissue in a centrifuge tube, soaking it in DPBS, inverting the centrifuge tube to clean the human sclerosing bone tissue, and incubating it with precooled BL buffer for pretreatment; tissue treatment and enzymatic hydrolysis: passing the incubated human sclerosing bone tissue and BL buffer through a cell screen, and rinsing the tissue 2-3 times with precooled PBS; transferring the human sclerosing bone tissue on the cell screen to a culture dish, adding BE solution, cutting the tissue into small pieces, and adding more BE solution; placing the culture dish in a 37°C constant temperature incubator, performing enzymatic hydrolysis for 15 minutes, and shaking the culture dish every 5 minutes; after the enzymatic hydrolysis is completed, transferring the enzymatic hydrolysis solution to a new centrifuge tube using a pipette; cell separation and collection: filtering the enzymatic hydrolysis solution using a cell screen, rinsing the cell screen with precooled DPBS containing 0.1% (m / v) BSA; centrifuging the filtrate in a horizontal centrifuge to collect cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a method for obtaining human sclerotinoma cells suitable for full transcriptome sequencing and its application. Background Art

[0002] The cellular components of bone tissue include osteoprogenitors, osteoblasts, osteocytes, and osteoclasts. Only osteocytes reside within bone tissue; the other three cell types are located at the edges of the bone tissue. After new bone matrix calcification, osteocytes are buried within the matrix's lacunas. Adjacent osteocytes communicate with each other, characterized by cytoplasmic processes extending into the bone matrix through osteocanaliculi. Osteocytes also connect with cells on the bone surface through the cytoplasmic processes and osteocanaliculi of adjacent bone surface cells. Thus, osteocytes form an extensive cellular communication network through their processes and the gap junctions between them. Transcriptome analysis of osteocytes is crucial for understanding the cellular regulatory networks of bone tissue. However, because osteocytes are embedded in the bone matrix, there is no effective way to obtain large numbers of free osteocytes. Consequently, methods specifically for preparing nuclear suspensions of hard bone tissue are lacking.

[0003] Single-cell sequencing (scRNA-seq) is a high-throughput technique for analyzing genetic information, including the genome, transcriptome, and epigenome, at the level of individual cells. It has become a state-of-the-art method for revealing the heterogeneity and complexity of RNA transcripts within individual cells, as well as the diverse cell types and functional components within tissues, organs, and organisms. Since its initial application in 2009, studies based on scRNA-seq have provided a wealth of information across diverse fields and have been widely applied to understanding cellular composition and interactions in humans, model animals, and plants. However, scRNA-seq primarily utilizes oligo-dT primers to initiate reverse transcription from the polyadenylated tail of mRNA, resulting in low sensitivity for detecting non-polyadenylated transcripts, such as lncRNAs, histone mRNAs, and rRNAs. These RNA molecules play a crucial role in regulating cellular function and status, but due to limitations in reverse transcription techniques, they cannot achieve "whole-transcriptome" single-cell profiling.

[0004] In summary, in order to solve the above problems, it is urgent to develop a simple and easy-to-operate single-cell whole transcriptome sequencing method suitable for hard bone tissue. Summary of the Invention

[0005] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide a single-cell whole transcriptome sequencing method suitable for human hard bone tissue. However, current technology is unable to complete the prerequisite of this scheme, that is, it is unable to complete the preparation of human hard bone tissue single-cell suspension. Therefore, in the present invention, the method for preparing human hard bone tissue single-cell suspension is also improved. The specific steps are "pretreatment of human hard bone tissue" and "obtaining human hard bone crude cell suspension".

[0006] Currently, existing single-cell transcriptome sequencing platforms, such as 10×Genomics and BD, are widely used in mammalian tissue research. This technology can complete RNA sequencing at the single-cell level, achieving qualitative and quantitative analysis of gene expression in individual cells in tissues. However, this technology can currently only detect intracellular coding RNA and a very small amount of long non-coding RNA. The reverse transcription process using oligodT as the main reverse transcription primer is fatal for most non-coding RNAs. In this case, a high-cost experiment is limited to detecting transcripts with polyadenylation, and cannot explore some non-coding RNAs that are important in gene expression regulation, signal transduction, metabolic regulation, and environmental response. Therefore, to address the shortcomings of the existing technology, the present invention, based on extensive in-depth research and experiments, provides a set of single-cell whole transcriptome sequencing experimental methods suitable for human hard bone tissue. This method is simple, easy to use, accurate, and has comprehensive detection information. It can be directly combined with the 10×Genomics and BD single-cell transcriptome sequencing processes, which can not only expand the application of single-cell transcriptome sequencing, but also obtain more detailed cell clustering effects and more comprehensive gene expression information with a smaller sequencing amount.

[0007] The present invention mainly proposes a concept of hard bone tissue processing, which is different from the current mechanical method plus enzymatic method that cannot complete the cell separation of human hard bone tissue. Through inorganic treatment, the inorganic components in the hard bone are decomposed to expose the extracellular matrix. In order to achieve this purpose, a BL buffer is proposed, in which the components are selected according to the conditions required for inorganic treatment. The BE solution is the same. After inorganic treatment, human hard bone tissue is no longer as hard as conventionally known. It does not require very high concentrations and strong enzymes for cell separation. Therefore, a suitable BE solution is proposed for hard bone tissue treated with BL buffer. The component is protease, which is commonly used in cell suspension preparation, but is a component that is not well known for this type of tissue. The human bone cell protectant optimizes the components of the fixative used in the fixation process to protect the integrity of the RNA structure, which is a necessary condition for completing single-cell whole transcriptome sequencing.

[0008] The present invention provides a pre-treatment method for preparing a cell suspension suitable for human hard bone tissue.

[0009] The pre-treatment method is to use BL buffer to perform inorganic treatment on hard bone before using protease to decompose the extracellular matrix.

[0010] 125 mg of human bone tissue was placed in an environment of 4-6°C and BL buffer was added for de-inorganization treatment to remove the hard matrix in the bone and expose the extracellular matrix to the buffer.

[0011] The BL buffer used is: 0.005-0.01M hydrochloric acid, 0.1%-0.2% (v / v) Tween 20 (Solarbio, T8220), 0.1%-0.2% (m / v) glutathione (Solarbio, G8180), 0.01M-0.02M TCEP (Thermo Fisher, T2556), and is prepared with deionized water. The pH is 5.5-6. The pH value is to provide an acidic environment to ensure the speed of inorganic removal. Too low a pH value will affect cell activity, and too high a pH value will affect the inorganic removal effect. Because the concentrations of hydrochloric acid and buffer components vary, the pH value can be between 5.5 and 6.

[0012] The role of the hydrochloric acid is to create an acidic environment, provide hydrogen ions to decompose inorganic substances in the hard bone, and expose the extracellular matrix.

[0013] The concentration of hydrochloric acid in the BL buffer is 0.005-0.01M, preferably 0.01M.

[0014] The Tween 20 is used to protect cell membrane proteins and prevent cell rupture caused by denaturation of cell membrane proteins during the de-inorganic treatment process.

[0015] The concentration of Tween 20 in the BL buffer is 0.1% to 0.2% (v / v), preferably 0.2% (v / v).

[0016] The role of the glutathione is to maintain the pH value of the buffer solution from changing significantly as the reaction proceeds, while protecting the cells from stress stimulation.

[0017] The glutathione concentration in the BL buffer is 0.1% to 0.2% (m / v), preferably 0.2% (m / v).

[0018] The tris(2-carboxyethyl)phosphine (TCEP) acts as a reducing agent to prevent cells from being damaged by oxidation.

[0019] The concentration of TCEP is 0.01M to 0.02M, preferably 0.02M TCEP.

[0020] The conditions for the bone de-inorganization treatment are 4° C. to 6° C. for 10 to 15 minutes, preferably 4° C. for 15 minutes.

[0021] The present invention also provides a method for preparing a single cell suspension of human bone tissue, and Figure 5The cell suspension treatment method for protecting cell nucleic acids corresponds to the specific implementation steps of using human bone protective agents to treat cells, and the application of the obtained single cell suspension in single cell whole transcriptome sequencing.

[0022] The present invention provides a method for preparing a single cell suspension from human hard bone tissue after being treated with BL buffer, which includes preparing a single cell suspension from human hard bone tissue after being treated with BL buffer using BE solution, and a method for purifying the crude human hard bone single cell suspension obtained by the above method.

[0023] The BE solution is prepared by dissolving 0.2% to 0.25% (m / v) collagenase I (Solarbio, C8140) and 0.2% to 0.25% (m / v) papain (Sigma, P4762) in 1×HBSS (Gibco, 14025092); preferably, the BE solution is prepared by dissolving 0.25% (m / v) collagenase I and 0.25% (m / v) papain in 1×HBSS (Gibco, 14025092).

[0024] The present invention proposes a solution suitable for single-cell whole transcriptome sequencing of human sclerotinoma cells. The BL buffer is a reagent component selected to achieve this purpose after the sclerotinoma tissue is pretreated in the present invention; for example, the two enzymes in the BE solution are used to dissociate soft tissue, and the combination of these two enzymes can obtain a cell suspension with higher viability and high yield after the hard tissue in the present invention is treated with BL buffer and the extracellular matrix is ​​exposed; the human sclerotinoma cell protective agent is optimized on the basis of fixed cells, and the components are optimized to maintain the integrity of the RNA structure, thereby achieving the ultimate experimental purpose.

[0025] The present invention also provides a specific method for preparing a human bone tissue single cell suspension using the BL buffer and the BE solution, comprising the following specific steps:

[0026] Step (1): Tissue washing: Place human bone tissue in a 50 mL centrifuge tube. Add 30 mL of DPBS (Gibco, 14190144) to the tube and place it in a 4-6°C environment. Soak for 3-5 minutes, slowly inverting the tube 3-5 times to remove the solution. Repeat the above steps once to complete tissue washing.

[0027] In step (1), the purpose of washing with DPBS (Gibco, 14190144) is to wash away free red blood cells and prevent the death of other target cells due to changes in oxygen content and pH in the buffer solution caused by the rupture of red blood cells during subsequent incubation.

[0028] In step (1), the cleaning conditions are preferably as follows: placing the tube in a 4°C environment and soaking for 5 minutes, during which the centrifuge tube is slowly inverted 5 times to remove the solution, and repeating the process once.

[0029] Step (2): Tissue pretreatment: Place human bone tissue in pre-cooled BL buffer and incubate at 4-6°C for 10-15 minutes, shaking horizontally for 30 seconds every 2-3 minutes.

[0030] In step (2), the BL buffer (tissue pretreatment solution) is used to decompose the hard inorganic components in the bone, thereby exposing the extracellular matrix and cells.

[0031] In step (2), the tissue pretreatment conditions are preferably incubated at 4°C for 15 minutes, with horizontal shaking every 3 minutes.

[0032] In step (2), the purpose of horizontally shaking the human bone tissue after treating it with the BL buffer is to accelerate the rate at which the BL buffer decomposes the inorganic components in the bone tissue and prevent the cell activity in the tissue from being affected by too long a time.

[0033] Step (3): After incubation, the human bone tissue and BL buffer were passed through a 100 μm cell sieve and the tissue was rinsed 2 to 3 times with pre-cooled PBS (Solarbio, P1020) buffer.

[0034] In step (3), the purpose of the 100 μm mesh filtration is to separate the processed hard bone tissue from the BL buffer. The 100 μm cell mesh can better retain the pretreated hard bone tissue. If the mesh aperture is too large, the tissue will pass through the mesh, resulting in a reduced cell yield. If the mesh aperture is too small, impurities will be retained on the mesh, which will have an adverse effect on the treatment of cells with the BE solution.

[0035] In step (3), the purpose of using pre-cooled PBS buffer to rinse the tissue is to wash away the BL buffer attached to the tissue to prevent it from affecting the pH value of the BE solution.

[0036] In step (3), the number of times the tissue is rinsed with pre-cooled PBS buffer is preferably 3 times.

[0037] Step (4): Transfer the human bone tissue on the cell sieve to a 70 mm culture dish, add 1 mL of BE solution to the culture dish, and use ophthalmic scissors to cut the bone tissue treated with BL buffer into 0.5 mm pieces. 3 Add BE solution to 4mL.

[0038] In step (4), the treated hard bone tissue is cut into pieces of 0.5 mm in size using ophthalmic scissors. 3 The purpose of the small blocks is to mechanically create more contact area between the BE solution and the extracellular matrix.

[0039] Step (5): Place the culture dish in a 37°C constant temperature incubator and allow the enzyme to hydrolyze for 15 minutes. Shake the culture dish every 5 minutes during the process. After the enzymatic hydrolysis is completed, use a pipette to transfer the enzymatic solution to a new centrifuge tube.

[0040] In step (5), the culture dish is placed in a 37°C constant temperature incubator to create a suitable environment for the BE solution to decompose the extracellular matrix.

[0041] In step (5), the purpose of shaking the culture dish every 5 minutes is to help the cells detach from the extracellular matrix and enter the BE solution.

[0042] Step (6): Filter the enzymatic hydrolysate (coarse cell suspension) obtained in step (5) using a 40 μm cell sieve.

[0043] In step (6), the purpose of cell filtration is to remove non-cellular substances with larger diameters from the coarse cell suspension.

[0044] Step (7): Rinse the cell screen with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin) (Miltenyi Biotec, 130-091-376).

[0045] In step (7), the purpose of flushing the screen is to reduce the loss of cells during the filtration process.

[0046] In step (7), the function of DPBS (Gibco, 14190144) containing 0.1 (m / v) BSA (bovine serum albumin) (Miltenyi Biotec, 130-091-376) is to maintain osmotic pressure and pH stability and protect cells.

[0047] Step (8): The collected filtrate is placed in a horizontal centrifuge for centrifugation to collect the cells.

[0048] In step (8), the centrifugal conditions for collecting cells are 300-500×g, 4-6°C, and 8-10 min. Preferably, the centrifugal conditions are 500×g, 4°C, and 10 min.

[0049] In step (8), the purpose of the centrifugation is to remove free nucleic acids, proteins and other cell contents in the cell suspension.

[0050] The present invention provides a human scleroblast protectant suitable for single-cell whole-transcriptome sequencing of human scleroblasts. Based on commonly used cell fixation methods, the invention improves the components to ensure the integrity of RNA in the cells, thereby meeting the requirements of single-cell whole-transcriptome sequencing for cells. The invention also includes the following specific steps:

[0051] Step (1): Add 3 mL of human sclerotin cell protective agent to the cell pellet obtained by centrifugation of the human sclerotin tissue single cell suspension, resuspend the cell pellet, and mix the suspension.

[0052] In step (1), the role of the human osteoblast protective agent is to effectively protect the integrity of RNA in a complex environment, keep the RNA structure intact, maintain the integrity of the enzyme recognition site during the treatment process, and ensure the reaction efficiency of the polyadenylation and reverse transcription processes.

[0053] In step (1), the pectin in the human scleroblast protection agent has the function of absorbing water and forming a hydration layer, thereby maintaining a moist environment within the cell and preventing dehydration and degradation of nucleic acids within the cell due to changes in the pH value and osmotic pressure of the extracellular environment.

[0054] In step (1), the components of the human scleroblast protectant are: 2-3% (v / v) paraformaldehyde PFA (Solarbio, P1110), 0.05-0.1% (m / v) pectin (Sigma, P7536), and ddH2O, preferably, 2% (v / v) PFA, 0.05% (m / v) pectin, and ddH2O.

[0055] Step (2): Place the centrifuge tube used to store cells in step (1) in a 4-6°C environment and let it stand for 8-10 minutes.

[0056] The incubation environment in step (2) is to provide suitable conditions for the human osteoblast protective agent.

[0057] In step (2), the incubation condition is 4-6°C for 8-10 minutes, preferably 4°C for 10 minutes.

[0058] Step (3): Add 5 mL of pre-cooled DPBS to the centrifuge tube, mix thoroughly by inverting the tube 4 to 5 times, and centrifuge in a horizontal centrifuge to enrich the cells.

[0059] In step (3), the purpose of adding pre-cooled DPBS and mixing by inversion is to slow down or terminate the reaction of the human osteocyte protective agent.

[0060] In step (3), the centrifugation conditions for centrifuging the enriched cells are 300-500×g, 4-6°C, and 8-10 min. Preferably, the centrifugation conditions are 500×g, 4°C, and 10 min.

[0061] The present invention provides a method for human osteocyte polyadenylation suitable for single-cell whole transcriptome sequencing of human osteocytes treated with a human osteocyte protective agent, comprising the following specific steps:

[0062] Step (1): Wash and centrifuge the cells twice with wash buffer.

[0063] In step (1), the washing buffer contains the following components (final concentration): 1× polyadenylate polymerase reaction buffer, 0.8 U / μl Protector RNase Inhibitor, and ddH2O.

[0064] In step (1), the centrifugation conditions are 300-500×g, 4-6°C, and 8-10 min. Preferably, the centrifugation conditions are 500×g, 4°C, and 10 min.

[0065] Step (2): Resuspend the cell pellet with polyadenylate polymerase mixture and mix well.

[0066] In step (2), the poly(A) polymerase mixture used comprises: 1× poly(A) polymerase reaction buffer, 24 U / μl poly(A) polymerase, 0.5 mM adenosine triphosphate (ATP), and 1.5 U / μl Protector RNase Inhibitor. Preferably, the poly(A) polymerase is a yeast-derived polymerase, the components of which are purchased from Thermo / 74225Z25KU, the poly(A) polymerase reaction buffer is a polymerase reaction buffer suitable for yeast-derived polymerases, the components of which are purchased from Thermo / 74225Z25KU, and ATP is purchased from Thermo / AM8110G.

[0067] In step (2), the amount of poly(A) polymerase mixture added was 1 mL.

[0068] In step (2), the mixing method is to slowly pipette and mix.

[0069] Step (3): incubate the suspension obtained in step (2) to complete the reaction.

[0070] In step (3), the incubation condition is 37° C. for 20 min.

[0071] In addition, the present invention also provides a single-cell whole transcriptome sequencing method applicable to human bone-derived cells obtained through the above-mentioned treatment, the specific steps of which are as follows:

[0072] Step (1): enrich the cells by centrifugation.

[0073] In step (1): the cells are human bone tissue cell suspension obtained by treating with BL buffer, BE solution, washing with washing buffer, and finally treating with polyadenylation enzyme mixture.

[0074] In step (1), the centrifugation conditions for washing cells are 300-500×g, 4-6°C, and 8-10 min. Preferably, the centrifugation conditions are 500×g, 4°C, and 10 min.

[0075] In step (1), the cleaning is performed 2 to 3 times, preferably twice.

[0076] Step (2): Wash the cells with DPBS containing bovine serum albumin.

[0077] Step (3): Resuspend the cells in DPBS containing bovine serum albumin.

[0078] Step (4): Calculate the cell concentration.

[0079] In step (2), the role of bovine serum albumin is to maintain the osmotic pressure and pH of the buffer solution and prevent the buffer environment from causing cell rupture.

[0080] In step (2) or (3), the content of bovine serum albumin is 0.1-0.2% (m / v), preferably 0.1% (m / v).

[0081] In step (4), the method for calculating the cell concentration was AO (acridine orange) (CA1143, Solarbio) staining, using a final concentration of 10 μg / mL.

[0082] Step (5): Adjust cell concentration.

[0083] In step (5), the cell concentration was adjusted to 1×10 6 pieces / mL.

[0084] Step (6): Single cell encapsulation and molecular experiments were performed according to the standard operating procedures of the Chromium Next GEM Single Cell 3′ GEM Kit v3.1 (Cat. No. 1000123) product of 10×Genomics (3′ Single Cell Transcriptome Sequencing Kit).

[0085] Step (7): Quality control of the library results and single-cell whole transcriptome sequencing and analysis.

[0086] In a specific embodiment, the specific steps of the method of the present invention are:

[0087] 1. Pretreatment of human bone tissue:

[0088] a. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0089] b. Prepare BL buffer: 0.005-0.01 M hydrochloric acid, 0.1-0.2% (v / v) Tween 20, 0.1-0.2% (m / v) glutathione, 0.01 M-0.02 M TCEP, using deionized water, pH = 5.5-6.

[0090] c. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4-6°C for 10-15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove inorganic matter. This differs from the pretreatment before suspension preparation in that the tissue is rinsed with DPBS to provide a suitable buffering environment for the BL buffer; the BL buffer treatment is for inorganic matter removal.

[0091] d. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0092] e. Transfer the tissue on the sieve obtained in step d to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0093] 2. Obtaining human bone cell suspension:

[0094] a. Prepare BE solution: 0.2-0.25% (m / v) collagenase I and 0.2-0.25% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092).

[0095] b. Add 1 mL of BE solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into 0.5 mm pieces. 3 Add BE solution to 4mL.

[0096] c. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0097] d. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0098] e. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0099] 3. Human osteocyte protection treatment:

[0100] a. Prepare human scleroblast protectant: 2-3% (v / v) PFA, 0.05-0.1% (m / v) pectin, ddH2O. Add 3 mL of PFA to the cell pellet obtained from human scleroblasts, mix well, and incubate at 4°C for 9-10 minutes.

[0101] b. Add 5 mL of pre-chilled DPBS to the centrifuge tube, mix thoroughly by inverting the tube five times, and centrifuge in a horizontal centrifuge to enrich the cells at 500 × g and 4°C for 10 min.

[0102] 4. Polyadenylation of non-coding RNA in human bone tissue cells:

[0103] a. Prepare wash buffer (final concentration): 1× poly(A) polymerase reaction buffer, 0.8 U / μl Protector RNase Inhibitor, ddH2O; prepare poly(A) polymerase mixture: 1× poly(A) polymerase reaction buffer, 24 U / μl poly(A) polymerase, 0.5 mM ATP, 1.5 U / μl Protector RNase Inhibitor.

[0104] b. Wash the cells twice with wash buffer. Centrifuge 3 mL of wash buffer at 500 × g for 10 min at 4°C.

[0105] c. Resuspend the human osteoblast pellet prepared in step b with 1 mL of polyadenylate polymerase mixture, pipette to mix thoroughly, and place in a 37°C incubator for 20 min.

[0106] d. After the reaction, centrifuge at 500×g for 10 min at 4°C to enrich human osteocytes and remove the enzyme mixture.

[0107] 5. Wash cells and perform single-cell whole transcriptome sequencing experiments:

[0108] a. Wash the cell pellet twice with 1 mL of 0.1% (m / v) bovine serum albumin in DPBS and centrifuge at 500 × g for 10 min at 4°C.

[0109] b. Resuspend the cells in 500 μL of 0.1% (m / v) bovine serum albumin (BSA)-containing DPBS, mix thoroughly by pipetting, and place the cells on ice until ready for use.

[0110] c. Use AO (acridine orange) at a final concentration of 10 μg / mL for cell staining, calculate the cell concentration using a fluorescence microscope with an excitation wavelength of 488 nm, and adjust the cell concentration to 1×106 cells / μL using 0.1% (m / v) bovine serum albumin in DPBS.

[0111] d. Refer to the standard operating procedures of 10×Genomics product Chromium Next GEM Single Cell 3′GEM Kit v3.1 (Cat. No. 1000123) for single-cell encapsulation and molecular experiments.

[0112] 6. Library result quality control

[0113] 7. Single-cell whole transcriptome sequencing and analysis

[0114] The present invention also provides applications of the above method in culturing human sclerotinoma cells, preparing human sclerotinoma single cell suspensions for single cell whole transcriptome sequencing, and studying human sclerotinoma cell division.

[0115] Compared with the prior art, the beneficial effects achieved by the present invention include: solving the problem of preparing single-cell suspensions of human hard bone tissue, completing the preparation of single-cell suspensions of human hard bone tissue, being suitable for hard bone tissue that is hard in texture and not easily broken by enzymatic hydrolysis, and avoiding the failure of cell suspension preparation caused by excessive damage to cells during the process of crushing tissue due to the hard texture of the tissue. Before polyadenylation of all non-coding RNA in a single cell, the cells are pretreated to protect the integrity of the RNA structure and increase the reaction rate. The capture of non-coding RNA can effectively increase the number of non-coding RNA (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) detected, improve the detection efficiency of non-coding RNA, and achieve the purpose of detecting the full transcription information of human hard bone tissue. In summary, the present invention provides a set of experimental processes suitable for single-cell whole transcriptome sequencing of human hard bone tissue, which can effectively complete the preparation of single-cell suspension of human hard bone tissue and detect multiple non-coding RNAs in cells, making up for the shortcomings of single-cell level research on human hard bone tissue, and providing a basis for in-depth understanding of the mechanisms in processes such as bone regeneration and bone repair, and for exploring unknown functional genes or regulatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0117] Figure 1 Flowchart of the method of the present invention.

[0118] Figure 2 This is the quality inspection result of cDNA 4150 in Example 1.

[0119] Figure 3 This is the quality inspection result of cDNA 4150 in Example 2.

[0120] Figure 4 This is the quality inspection result of library 4150 in Example 1.

[0121] Figure 5 This is the quality inspection result of library 4150 in Example 2.

[0122] Figure 6 This is a diagram of the RNA structure detected in Example 1.

[0123] Figure 7 This is a diagram of the RNA structure detected in Example 2.

[0124] Figure 8 For comparison with the enzymatic hydrolysis results of Example 3, fluorescence microscopy revealed that the proportion of living cells was extremely low.

[0125] Figure 9 For comparison with the enzymatic hydrolysis results of Example 15, fluorescence microscopy revealed a high proportion of live cells.

[0126] Figure 10 This is a fluorescence microscopy image of the human osteocyte suspension in Example 1.

[0127] Figure 11 This is a fluorescence microscopy image of the human osteocyte suspension in Example 2. DETAILED DESCRIPTION

[0128] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0129] The present invention discloses a sample preparation scheme suitable for single-cell whole-transcriptome sequencing of human sclerotin. The method includes a method for preparing a human sclerotin cell suspension suitable for single-cell whole-transcriptome sequencing; a human sclerotin cell protectant that effectively protects the integrity of RNA in complex environments, maintains the integrity of the RNA structure, maintains the integrity of enzyme recognition sites during processing, and ensures the reaction efficiency of polyadenylation and reverse transcription. The method also includes a method for RNA polyadenylation in human sclerotin cells that can effectively polyadenylate non-coding RNA in human sclerotin cell samples, simplifying the detection of single-cell non-coding RNA.

[0130] Example 1

[0131] 1. Pretreatment of human bone tissue:

[0132] a. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0133] b. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0134] c. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove inorganic matter.

[0135] d. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0136] e. Transfer the tissue on the sieve obtained in step (d) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0137] 2. Obtaining human bone cell suspension:

[0138] a. Prepare BE solution: 0.25% (m / v) collagenase I and 0.25% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092).

[0139] b. Add 1 mL of BE solution to the culture dish. Use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3. Add more BE solution to 4 mL.

[0140] c. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0141] d. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0142] e. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0143] 3. Human osteoblast pretreatment:

[0144] a. Add 3 mL of human osteoblast cell protective agent to the cell pellet obtained from human sclerotin tissue, mix the suspension, and incubate at 4°C for 9-10 minutes.

[0145] b. Add 5 mL of pre-chilled DPBS to the centrifuge tube, mix thoroughly by inverting the tube five times, and centrifuge in a horizontal centrifuge to enrich the cells at 500 × g and 4°C for 10 min.

[0146] 4. Polyadenylation of non-coding RNA in human bone tissue cells:

[0147] a. Prepare wash buffer (final concentration): 1× poly(A) polymerase reaction buffer, 0.8 U / μl Protector RNase Inhibitor, ddH2O; prepare poly(A) polymerase mixture: 1× poly(A) polymerase reaction buffer, 24 U / μl poly(A) polymerase, 0.5 mM ATP, 1.5 U / μl Protector RNase Inhibitor.

[0148] b. Wash the cells twice with wash buffer. Centrifugation conditions for washing are: 3 mL of wash buffer, 500 × g, 4°C, 10 min.

[0149] c. Resuspend the human osteoblast pellet from step (b) with 1 mL of polyadenylate polymerase mixture, pipette to mix thoroughly, and place in a 37°C incubator for 20 min.

[0150] d. After the reaction, centrifuge at 500×g for 10 min at 4°C to enrich human osteocytes and remove the enzyme mixture.

[0151] 5. Wash cells and perform single-cell whole transcriptome sequencing experiments:

[0152] a. Wash the cell pellet twice with 1 mL of 0.1% (m / v) bovine serum albumin in DPBS and centrifuge at 500 × g for 10 min at 4°C.

[0153] b. Resuspend the cells in 500 μL of 0.1% (m / v) bovine serum albumin (BSA)-containing DPBS, mix thoroughly by pipetting, and place the cells on ice until ready for use.

[0154] c. Use AO (acridine orange) at a final concentration of 10 μg / mL for cell staining, calculate the cell concentration using a fluorescence microscope with an excitation wavelength of 488 nm, and adjust the cell concentration to 1×106 cells / μL using 0.1% (m / v) bovine serum albumin in DPBS.

[0155] d. Refer to the standard operating procedures of 10×Genomics product Chromium Next GEM Single Cell 3′GEM Kit v3.1 (Cat. No. 1000123) for single-cell encapsulation and molecular experiments.

[0156] 6. Library result quality control

[0157] 7. Single-cell whole transcriptome sequencing and analysis

[0158] Example 2

[0159] 1. Pretreatment of human bone tissue:

[0160] a. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 6°C environment. Let it soak for 3 minutes. During this time, slowly invert the tube 3 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0161] b. Prepare BL buffer: 0.005 M hydrochloric acid, 0.1% Tween 20, 0.1% glutathione, 0.01 M TCEP, using deionized water, pH = 6.0.

[0162] c. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 6°C for 13 minutes, shaking horizontally for 30 seconds every 2 minutes to remove inorganic matter.

[0163] d. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0164] e. Transfer the tissue on the sieve obtained in step (d) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0165] 2. Obtaining human bone cell suspension:

[0166] a. Prepare BE solution: 0.2% (m / v) collagenase I and 0.2% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092).

[0167] b. Add 1 mL of BE solution to the culture dish. Use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3. Add more BE solution to 4 mL.

[0168] c. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 20 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0169] d. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0170] e. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0171] 3. Human osteoblast pretreatment:

[0172] a. Add 3 mL of human osteoblast cell protective agent to the cell pellet obtained from human sclerotin tissue, mix the suspension, and incubate at 4°C for 9-10 minutes.

[0173] b. Add 5 mL of pre-chilled DPBS to the centrifuge tube, mix thoroughly by inverting the tube five times, and centrifuge in a horizontal centrifuge to enrich the cells at 500 × g and 4°C for 10 min.

[0174] 4. Polyadenylation of non-coding RNA in human bone tissue cells:

[0175] a. Prepare wash buffer (final concentration): 1× poly(A) polymerase reaction buffer, 0.8 U / μl Protector RNase Inhibitor, ddH2O; prepare poly(A) polymerase mixture: 1× poly(A) polymerase reaction buffer, 24 U / μl poly(A) polymerase, 0.5 mM ATP, 1.5 U / μl Protector RNase Inhibitor.

[0176] b. Wash the cells twice with wash buffer. Centrifugation conditions for washing are: 3 mL of wash buffer, 500 × g, 4°C, 10 min.

[0177] c. Resuspend the human osteoblast pellet from step (b) with 1 mL of polyadenylate polymerase mixture, pipette to mix thoroughly, and place in a 37°C incubator for 20 min.

[0178] d. After the reaction, centrifuge at 500×g for 10 min at 4°C to enrich human osteocytes and remove the enzyme mixture.

[0179] 5. Wash cells and perform single-cell whole transcriptome sequencing experiments:

[0180] a. Wash the cell pellet twice with 1 mL of 0.1% (m / v) bovine serum albumin in DPBS and centrifuge at 500 × g for 10 min at 4°C.

[0181] b. Resuspend the cells in 500 μL of 0.1% (m / v) bovine serum albumin (BSA)-containing DPBS, mix thoroughly by pipetting, and place the cells on ice until ready for use.

[0182] c. Use AO (acridine orange) at a final concentration of 10 μg / mL for cell staining, calculate the cell concentration using a fluorescence microscope with an excitation wavelength of 488 nm, and adjust the cell concentration to 1×106 cells / μL using 0.1% (m / v) bovine serum albumin in DPBS.

[0183] d. Refer to the standard operating procedures of 10×Genomics product Chromium Next GEM Single Cell 3′GEM Kit v3.1 (Cat. No. 1000123) for single-cell encapsulation and molecular experiments.

[0184] 6. Library result quality control

[0185] 7. Single-cell whole transcriptome sequencing and analysis

[0186] Comparative Example 1

[0187] In this comparative example, approximately 150 mg of human bone tissue cells were isolated by grinding in a mortar for 15 minutes.

[0188] The results of cell quantity and quality assessment are shown in Table 2.

[0189] Comparative Example 2

[0190] In this comparative example, approximately 150 mg of human bone tissue cells were separated using the Bone Marrow Dissociation Kit of Miltenyi Biotec.

[0191] The experiment was completed according to the SOP, and the results of cell quantity and quality assessment are shown in Table 2.

[0192] Comparative Example 3

[0193] This comparative example uses Miltenyi Biotec's MACS Tissue Dissociation Kits to separate approximately 150 mg of human bone tissue cells.

[0194] The experiment was completed according to the SOP, and the results of cell quantity and quality assessment are shown in Table 2.

[0195] Comparative Example 4

[0196] In this comparative example, approximately 150 mg of human scleral bone tissue cells were isolated using a Mouse Skeletal Tissue Dissociation Kit.

[0197] The experiment was completed according to the SOP, and the results of cell quantity and quality assessment are shown in Table 2.

[0198] Comparative Example 5

[0199] In this comparative example, approximately 150 mg of human bone tissue cells were separated using a Mouse Skeletal Tissue Dissociation Kit (Stemcell Technologies).

[0200] The experiment was completed according to the SOP, and the enzymatic hydrolysis time was extended to 12 h. The results of cell quantity and quality assessment are shown in Table 2.

[0201] Comparative Example 6

[0202] This comparative example uses MACS Tissue Dissociation Kits to separate approximately 150 mg of human bone tissue cells.

[0203] The experiment was completed according to the SOP, and the enzymatic hydrolysis time was extended to 12 h. The results of cell quantity and quality assessment are shown in Table 2.

[0204] Comparative Example 7

[0205] In this comparative example, approximately 150 mg of human scleral bone tissue cells were isolated using a Mouse Skeletal Tissue Dissociation Kit.

[0206] The experiment was completed according to the SOP, and the enzymatic hydrolysis time was extended to 12 h. The results of cell quantity and quality assessment are shown in Table 2.

[0207] Comparative Example 8

[0208] In this comparative example, a cell suspension of approximately 150 mg of human bone tissue sample was prepared with reference to "Single-cell RNA-seq of human articular chondrocytes in osteoarthritis reveals disease-associated molecular signatures".

[0209] 1. Tissue processing: mince or chop the bone sample using scissors or a scalpel.

[0210] 2. Enzyme digestion: Use a digestion solution containing collagenase II and DNase I for enzymatic digestion.

[0211] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0212] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0213] 5. Cell quantity and quality assessment: 0.04% (m / v) trypan blue was used to stain the cells for evaluation of cell quantity and quality. The results are shown in Table 2.

[0214] Comparative Example 9

[0215] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0216] After treating approximately 150 mg of human bone tissue with BL buffer, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0217] Comparative Example 10

[0218] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 2 (0.01 M nitric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0219] After treating approximately 150 mg of human bone tissue with BL buffer 2, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0220] Comparative Example 11

[0221] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 3 (0.01 M perchloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0222] After treating approximately 150 mg of human bone tissue with BL buffer 3, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0223] Comparative Example 12

[0224] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 4 (0.01 M hydroiodic acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0225] After treating approximately 150 mg of human bone tissue with BL buffer 4, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0226] Comparative Example 13

[0227] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 5 (0.01 M hydrobromic acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0228] After treating approximately 150 mg of human bone tissue with BL buffer 5, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0229] Comparative Example 14

[0230] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 6 (0.005 M sulfuric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0231] After treating approximately 150 mg of human bone tissue with BL buffer 6, the liquid was removed, the treated tissue state was observed, and the tissue was weighed. The results are shown in Table 3.

[0232] Comparative Example 15

[0233] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0234] 1. Tissue processing: About 150 mg of human bone sample treated with BL Buffer 7 was minced or chopped using scissors or a scalpel.

[0235] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0236] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0237] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0238] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0239] The results are shown in Table 4.

[0240] Comparative Example 16

[0241] In this comparative example, BL buffer 7 (0.01 M hydrochloric acid, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) was used to pretreat approximately 150 mg of human bone tissue at 4° C. for 15 min.

[0242] 1. Tissue processing: About 150 mg of human bone sample treated with BL Buffer 7 was minced or chopped using scissors or a scalpel.

[0243] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0244] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0245] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0246] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0247] The results are shown in Table 4.

[0248] Comparative Example 17

[0249] In this comparative example, BL buffer 8 (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.02 M TCEP, prepared with deionized water, pH = 5.5) was used to pretreat approximately 150 mg of human bone tissue at 4° C. for 15 min.

[0250] 1. Tissue processing: About 150 mg of human bone sample treated with BL Buffer 8 was minced or chopped using scissors or a scalpel.

[0251] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0252] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0253] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0254] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0255] The results are shown in Table 4.

[0256] Comparative Example 18

[0257] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 9 (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, prepared with deionized water, pH = 5.5) at 4°C for 15 min.

[0258] 1. Tissue processing: About 150 mg of human bone sample treated with BL Buffer 9 was minced or chopped using scissors or a scalpel.

[0259] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0260] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0261] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0262] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0263] The results are shown in Table 4.

[0264] Comparative Example 19

[0265] In this comparative example, BL buffer 10 (0.002 M hydrochloric acid, 0.05% Tween 20, 0.05% glutathione, 0.005 M TCEP, prepared with deionized water) was used to pretreat approximately 150 mg of human bone tissue at 4° C. for 15 min.

[0266] 1. Tissue processing: About 150 mg of human bone sample was treated with BL buffer 10 and minced or cut into pieces using scissors or a scalpel.

[0267] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0268] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0269] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0270] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0271] The results are shown in Table 5.

[0272] Comparative Example 20

[0273] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer 11 (0.015 M hydrochloric acid, 0.3% (v / v) Tween 20, 0.3% (m / v) glutathione, 0.03 M TCEP, prepared with deionized water) at 4° C. for 15 min.

[0274] 1. Tissue processing: About 150 mg of human bone sample was treated with BL buffer 11 and minced or chopped using scissors or a scalpel.

[0275] 2. Enzyme digestion: Use digestion solution containing collagenase I for enzymatic digestion.

[0276] 3. Mechanical dispersion: After enzymatic digestion, the enzymatically digested sample is homogenized.

[0277] 4. Treatment of single-cell suspension: After mechanical dispersion, filter the single-cell suspension using a 40 μm cell sieve; centrifuge the filtrate and resuspend the cell pellet in DPBS.

[0278] 5. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0279] The results are shown in Table 5.

[0280] Comparative Example 21

[0281] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 8 min.

[0282] The remaining steps are the same as those in Comparative Example 20.

[0283] The results are shown in Table 6.

[0284] Comparative Example 22

[0285] In this comparative example, approximately 150 mg of human bone tissue was pretreated using BL buffer (0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5) at 4°C for 17 min.

[0286] The remaining steps are the same as those in Comparative Example 20.

[0287] The results are shown in Table 6.

[0288] Comparative Example 23

[0289] In this comparative example, 0.2% (m / v) collagenase II was used at 37 degrees Celsius for 15 minutes to separate tissue cells.

[0290] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0291] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0292] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0293] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0294] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0295] 6. Prepare enzymatic solution for later use: Dissolve 0.25% (m / v) collagenase II in 1× HBSS (Gibco, 14025092).

[0296] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0297] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0298] 9. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0299] 10. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0300] 11. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0301] The results are shown in Table 7.

[0302] Comparative Example 24

[0303] In this comparative example, 0.1% (m / v) bovine serum albumin, 0.2% (m / v) collagenase I, and HBSS (containing calcium and magnesium) were used for tissue cell separation at 37 degrees Celsius for 15 minutes.

[0304] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0305] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0306] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0307] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0308] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0309] 6. Prepare enzymatic solution: 0.1% (m / v) bovine serum albumin, 0.2% (m / v) collagenase I, dissolved in 1× HBSS (Gibco, 14025092).

[0310] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0311] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0312] 9. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0313] 10. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0314] 11. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0315] The results are shown in Table 7.

[0316] Comparative Example 25

[0317] In this comparative example, 0.2% (m / v) collagenase I, 75 U / mL DNase I, and HBSS (containing calcium and magnesium) were used for tissue cell separation at 37 degrees Celsius for 15 minutes.

[0318] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0319] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0320] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0321] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0322] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0323] 6. Prepare enzymatic solution: 0.2% (m / v) collagenase I, 75 U / mL DNase I, dissolved in 1× HBSS (Gibco, 14025092).

[0324] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0325] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0326] 9. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0327] 10. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0328] 11. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0329] The results are shown in Table 7.

[0330] Comparative Example 26

[0331] In this comparative example, 0.2% (m / v) collagenase I and HBSS (containing calcium and magnesium) were used at 37 degrees Celsius for 15 minutes to separate tissue cells, and then 0.25% (m / v) trypsin was used for treatment for 3 minutes.

[0332] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0333] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0334] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0335] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0336] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0337] 6. Prepare enzymatic solution: 0.2% (m / v) collagenase I, dissolved in 1× HBSS (Gibco, 14025092).

[0338] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0339] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0340] 9. Add 0.25% (m / v) pancreatin to the enzymatic solution obtained in step (8) and treat for 3 minutes

[0341] 10. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0342] 11. Collect the cells by centrifugation at 500×g, 4°C, and 10 min.

[0343] 12. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0344] The results are shown in Table 7.

[0345] Comparative Example 27

[0346] In this comparative example, 0.25% (m / v) papain and 0.25% (m / v) collagenase I were used in HBSS (containing calcium and magnesium) at 37 degrees Celsius for 15 minutes to separate tissue cells.

[0347] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0348] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0349] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0350] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0351] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0352] 6. Prepare enzymatic solution: 0.25% (m / v) papain and 0.25% (m / v) collagenase I, dissolved in 1× HBSS (Gibco, 14025092).

[0353] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0354] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0355] 9. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0356] 10. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0357] 11. Assessment of cell quantity and quality: 0.04% (m / v) trypan blue was used for cell staining to assess cell quantity and quality.

[0358] The results are shown in Table 7.

[0359] Comparative Example 28

[0360] In this comparative example, 0.25% (m / v) papain and 0.25% (m / v) collagenase I were used in HBSS (without calcium and magnesium) at 37 degrees Celsius for 15 minutes to separate tissue cells.

[0361] The remaining operations are the same as those in Comparative Example 27.

[0362] The results are shown in Table 7

[0363] Comparative Example 29

[0364] In this comparative example, 0.25% (m / v) papain and 0.25% (m / v) collagenase I were used in DPBS at 37 degrees Celsius for 15 minutes to separate tissue cells.

[0365] The remaining operations are the same as those in Comparative Example 27.

[0366] The results are shown in Table 7

[0367] Comparative Example 30

[0368] In this comparative example, BE solution 2 (0.1% (m / v) collagenase I, 0.1% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092)) was used for tissue cell separation at 37 degrees Celsius for 15 minutes.

[0369] The remaining operations are the same as those in Comparative Example 27.

[0370] The results are shown in Table 8

[0371] Comparative Example 31

[0372] In this comparative example, BE solution 3 (0.3% (m / v) collagenase I, 0.3% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092)) was used for tissue cell separation at 37 degrees Celsius for 15 minutes.

[0373] The remaining operations are the same as those in Comparative Example 27.

[0374] The results are shown in Table 8

[0375] Comparative Example 32

[0376] In this comparative example, 0.25% (m / v) collagenase I and 0.25% (m / v) papain were used in 1×HBSS (Gibco, 14025092) at 37 degrees Celsius for 10 minutes to separate tissue cells.

[0377] The remaining operations are the same as those in Comparative Example 27.

[0378] The results are shown in Table 9

[0379] Comparative Example 33

[0380] In this comparative example, 0.25% (m / v) collagenase I and 0.25% (m / v) papain were used in 1×HBSS (Gibco, 14025092) at 37 degrees Celsius for 25 minutes to separate tissue cells.

[0381] The remaining operations are the same as those in Comparative Example 27.

[0382] The results are shown in Table 9

[0383] Comparative Example 34

[0384] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and the obtained cells were fixed with 3% paraformaldehyde at 4° C. for 10 minutes.

[0385] 1. Place the bone tissue in a 50mL centrifuge tube. Add 30mL of DPBS to the tube and place it in a 4°C environment. Let it soak for 5 minutes. During this time, slowly invert the tube 5 times to remove the solution. Repeat this step once to complete tissue cleaning.

[0386] 2. Prepare BL buffer: 0.01 M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, using deionized water, pH = 5.5.

[0387] 3. Place approximately 150 mg of human bone tissue in 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to remove the inorganic matter.

[0388] 4. After incubation, pass the bone tissue and BL buffer through a 100 μm cell sieve and rinse the tissue three times with pre-cooled PBS buffer.

[0389] 5. Transfer the tissue on the sieve obtained in step (4) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0390] 6. Prepare enzymatic solution: 0.25% (m / v) papain and 0.25% (m / v) collagenase I, dissolved in 1× HBSS (Gibco, 14025092).

[0391] 7. Add 1 mL of enzymatic hydrolysis solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; add more enzymatic hydrolysis solution to 4 mL.

[0392] 8. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0393] 9. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0394] 10. Collect the cells by centrifugation at 500×g and 4°C for 10 min.

[0395] 11. Resuspend the cells in step (10) with 3% paraformaldehyde, pipette to mix, and fix at 4°C for 10 min.

[0396] 12. RNA extraction quality control: TRIZOL precipitation method was used to extract cellular RNA, and the RNA quality was assessed using the Agilent 2100 Bioanalyzer.

[0397] The results are shown in Table 10.

[0398] Comparative Example 35

[0399] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and fixed with methanol at -20°C for 20 minutes.

[0400] The remaining steps are the same as those in Comparative Example 33.

[0401] The results are shown in Table 10.

[0402] Comparative Example 36

[0403] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and RNAlater was used to fix the tissue at 4° C. for 10 minutes.

[0404] The remaining steps are the same as those in Comparative Example 33.

[0405] The results are shown in Table 10.

[0406] Comparative Example 37

[0407] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and fixed with 3% paraformaldehyde and 4 U / ml RNase inhibitor at 4° C. for 10 minutes.

[0408] The remaining steps are the same as those in Comparative Example 33.

[0409] The results are shown in Table 10.

[0410] Comparative Example 38

[0411] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and fixed with 3% paraformaldehyde and 0.02% pectin at 4° C. for 10 minutes.

[0412] The remaining steps are the same as those in Comparative Example 33.

[0413] The results are shown in Table 10.

[0414] Comparative Example 39

[0415] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and fixed with 3% paraformaldehyde and 0.02% agarose at 4° C. for 10 minutes.

[0416] The remaining steps are the same as those in Comparative Example 33.

[0417] The results are shown in Table 10.

[0418] Comparative Example 40

[0419] In this comparative example, human sclerotin tissue was treated with BL buffer and BE solution in sequence, and human sclerotin cell protective agent 2 (1% (v / v) PFA, 0.03% (m / v) pectin, ddH2O) was used for fixation at 4°C for 10 min.

[0420] The remaining steps are the same as those in Comparative Example 33.

[0421] The results are shown in Table 11.

[0422] Comparative Example 41

[0423] In this comparative example, human sclerotin tissue was treated with BL buffer and BE solution in sequence, and human sclerotin cell protective agent 3 (4% (v / v) PFA, 0.3% (m / v) pectin, ddH2O) was used for fixation at 4°C for 10 min.

[0424] The remaining steps are the same as those in Comparative Example 33.

[0425] The results are shown in Table 11.

[0426] Comparative Example 42

[0427] In this comparative example, human sclerotin tissue was treated with BL buffer and BE solution in sequence, and fixed with human sclerotin cell protective agents 3% (v / v) PFA, 0.1% (m / v) pectin, and ddH2O at 4°C for 7 minutes.

[0428] The remaining steps are the same as those in Comparative Example 33.

[0429] The results are shown in Table 12.

[0430] Comparative Example 43

[0431] In this comparative example, human bone tissue was treated with BL buffer and BE solution in sequence, and fixed with human bone cell protective agents 3% (v / v) PFA, 0.1% (m / v) pectin, and ddH2O at 4°C for 15 minutes.

[0432] The remaining steps are the same as those in Comparative Example 33.

[0433] The results are shown in Table 12.

[0434] Comparative Example 44

[0435] This comparative example uses BL buffer to treat mouse hard bone tissue

[0436] 1. Take mouse femur tissue (~200 mg) and place it in a 70 μm culture dish.

[0437] 2. Add 5 mL of BL buffer and incubate at 4°C for 15 minutes, shaking horizontally for 30 seconds every 3 minutes to carry out inorganic removal reaction.

[0438] 3. Observe and detect tissue cell status

[0439] The experimental results are shown in Table 13.

[0440] Comparative Example 45

[0441] This comparative example uses BL buffer and BE solution to treat human skeletal muscle tissue

[0442] 1. Take human skeletal muscle tissue (approximately 150 mg) and place it in a 70 μm culture dish.

[0443] 2. Add 5 mL of BL buffer and incubate at 4°C for 15 min, shaking horizontally for 30 s every 3 min to remove inorganic matter.

[0444] 3. Transfer the tissue on the sieve obtained in step (2) to a 70 mm diameter culture dish and keep the tissue on ice during the experiment.

[0445] 4. Add 1 mL of BE solution to the culture dish and use ophthalmic scissors to cut the tissue treated with BL buffer into small pieces of 0.5 mm3 in size; then add more BE solution to 4 mL.

[0446] 5. Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0447] 6. Filter the digestion solution obtained in the above step using a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0448] 7. Collect the cells by centrifugation at 500 × g for 10 min at 4°C. Reselect the cells with 200 μL of pre-cooled DPBS containing 0.1% (m / v) BSA (bovine serum albumin). Detect the cell status using 0.04% (m / v) trypan blue staining.

[0449] The experimental results are shown in Table 13.

[0450] Comparative Example 46

[0451] This comparative example uses human osteoblast protective agents to treat human PBMC cells

[0452] 1. Take human PBMC cells (1×105) and place them in a centrifuge tube for centrifugation and enrichment.

[0453] 2. Reselect the cell pellet from step (1) using 1 mL of human osteoblast protective agent, mix thoroughly by pipetting, and incubate at 4°C for 10 min.

[0454] 3. Observe and detect the cell status, and extract cell RNA for detection using Agilent 2100 Bioanalyzer.

[0455] The experimental results are shown in Table 14.

[0456] Table 1 Examples 1-2

[0457]

[0458] The present invention provides a single-cell whole-transcriptome sequencing experimental process suitable for human sclerotin tissue, which includes a buffer solution (BL buffer) suitable for pretreatment of human sclerotin tissue cells before isolation, a reagent solution (BE solution) suitable for cell separation of human sclerotin tissue after BL buffer treatment, a polyadenylation pretreatment method suitable for human sclerotin tissue cells, a polyadenylation treatment method suitable for cells in human sclerotin tissue, and a single-cell whole-transcriptome sequencing process. All of the above methods described in the present invention can be directly integrated into the conventional 10X Genomics Chromium NextGEM Automated Single Cell 3'cDNAKit v3.1 process, filling the gap in the current application of high-throughput single-cell whole-transcriptome sequencing technology for human sclerotin tissue and contributing to a deeper understanding of the mechanisms of bone regeneration and repair.

[0459] The present invention relates to a BL buffer solution suitable for preparing single cell suspension of human hard bone tissue, and a method for using the same. The BL buffer solution comprises: 0.005-0.01M hydrochloric acid, 0.1%-0.2% Tween 20, 0.1%-0.2% glutathione, 0.01M-0.02M TCEP, prepared with deionized water, pH = 5.5-6; a BE solution suitable for preparing a single-cell suspension of human hard bone tissue and a method for preparing a single-cell suspension of human hard bone tissue, the BE solution ingredients are: 0.2%-0.25% (m / v) collagenase I; 0.2%-0.25% (m / v) papain dissolved in 1×HBSS (Gibco, 14025092); a cell protectant suitable for human hard bone tissue cells and a method for using the same, the human hard bone tissue cell protectant ingredients are: 2-3% (v / v) PFA, 0.05-0.1% (m / v) pectin, ddH2O; a method for polyadenylation of human hard bone tissue cells and an application for whole-transcriptome sequencing of single cells of human hard bone tissue. The present invention is designed to be suitable for whole-transcriptome sequencing of single cells of human hard bone tissue, specifically:

[0460] 1) Place the bone tissue in a 50 mL centrifuge tube. Add 30 mL of DPBS and incubate at 4°C for 5 minutes. Gently invert the tube 5 times to remove the solution. Repeat this procedure once to complete tissue cleaning.

[0461] 2) Prepare BL buffer: 0.005-0.01 M hydrochloric acid, 0.1-0.2% Tween 20, 0.1-0.2% glutathione, 0.01 M-0.02 M TCEP, using deionized water, pH = 5.5-6.

[0462] 3) Approximately 150 mg of human bone tissue was placed in 5 mL of BL buffer and incubated at 4-6°C for 10-15 minutes, with horizontal shaking for 30 seconds every 3 minutes to allow for inorganic desorption.

[0463] 4) After incubation, the bone tissue and BL buffer were passed through a 100 μm cell sieve and rinsed three times with pre-cooled PBS buffer.

[0464] 5) The tissue on the sieve obtained in 4) above was transferred to a 70 mm diameter culture dish and kept on ice during the experiment.

[0465] 6) Prepare BE solution: 0.2-0.25% (m / v) collagenase I and 0.2-0.25% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092).

[0466] 7) Add 1 mL of BE solution to the culture dish and use ophthalmic scissors to cut the hard bone tissue treated with BL buffer into small pieces of 0.5 mm3; then add more BE solution to 4 mL.

[0467] 8) Place the culture dish in a 37°C incubator and allow the enzyme to digest for 15-20 minutes. Shake the dish every 5 minutes. After digestion is complete, transfer the solution to a new centrifuge tube using a pipette.

[0468] 9) Filter the digestion solution obtained in the above step through a 40 μm cell sieve, rinse the cell sieve with 5 mL of pre-cooled DPBS containing 0.1 (m / v) BSA (bovine serum albumin), and collect the filtrate.

[0469] 10) Collect the cells by centrifugation at 500×g for 10 min at 4° C., and remove the supernatant.

[0470] 11) Prepare human osteoblast protective agent: 2-3% (v / v) PFA, 0.05-0.1% (m / v) pectin, ddH2O.

[0471] 12) Add 3 mL of human sclerotin cell protective agent to the cell pellet obtained from human sclerotin tissue in 10) above, mix the suspension, and incubate at 4° C. for 9-10 minutes.

[0472] 13) Add 5 mL of pre-chilled DPBS to the centrifuge tube, mix thoroughly by inverting the tube five times, and centrifuge in a horizontal centrifuge to enrich the cells at 500 × g, 4°C, and 10 min.

[0473] 14) Prepare wash buffer (final concentration): 1× poly(A) polymerase reaction buffer, 0.8 U / μl Protector RNase Inhibitor, ddH2O; prepare poly(A) polymerase mixture: 1× poly(A) polymerase reaction buffer, 24 U / μl poly(A) polymerase, 0.5 mM ATP, 1.5 U / μl Protector RNase Inhibitor.

[0474] 15) Wash the cells twice with wash buffer. Washing centrifugation conditions are: 3 mL of wash buffer, 500 × g, 4°C, 10 min.

[0475] 16) Resuspend the human osteoblast pellet from 15) above with 1 mL of polyadenylate polymerase mixture, pipette to mix thoroughly, and place in a 37°C incubator for 20 minutes.

[0476] 17) After the reaction, the human osteoblasts were enriched by centrifugation at 500×g and 4°C for 10 min, and the enzyme mixture was removed.

[0477] 18) Wash the cell pellet twice with 1 mL of 0.1% (m / v) bovine serum albumin in DPBS and centrifuge at 500×g for 10 min at 4°C.

[0478] 19) Resuspend the cells in 500 μL of 0.1% (m / v) bovine serum albumin (BSA)-containing DPBS, mix thoroughly by pipetting, and place the cells on ice until ready for use.

[0479] 20) Cells were stained with AO (acridine orange) at a final concentration of 10 μg / mL, and the cell concentration was calculated using a fluorescence microscope with an excitation wavelength of 488 nm. The cell concentration was adjusted to 1 × 10 6 cells / μL using 0.1% (m / v) bovine serum albumin in DPBS.

[0480] 21) Single-cell encapsulation and molecular experiments were performed according to the standard operating procedures of the Chromium Next GEM Single Cell 3′ GEM Kit v3.1 (Cat. No. 1000123) product of 10× Genomics.

[0481] 22) Library quality control

[0482] 23) Single-cell whole transcriptome sequencing and analysis

[0483] In order to design a method and process for single-cell whole transcriptome sequencing experiments of human hard bone tissue, the present invention attempts to design a polyadenylation step based on the single-cell transcriptome. In view of the high hardness and high strength of human hard bone tissue, a series of explorations were conducted on the preparation method of single-cell suspension. Based on the obtained single-cell suspension, a series of comparative experiments were carried out, using indicators such as cell number in cell suspension, cell viability, cell number after polyadenylation treatment, cell viability, cDNA quality, library quality, and non-coding RNA capture efficiency to analyze the effect of polyadenylation on the quality of the cDNA. The above method was evaluated using the following detection criteria: 100,000 to approximately 150,000 cells were obtained per 150 mg of hard bone tissue, with a cell viability greater than 90%; 50,000 to 70,000 intact cells were obtained after polyadenylation treatment, all of which could be stained with AO dye; cDNA fragment length was 900 to 1200 bp, with a concentration greater than 10 ng / μL; library fragment length was 400 to 450 bp, with a concentration greater than 40 ng / μL; and the cDNA library could detect low-abundance non-coding RNA.

[0484] In order to obtain an experimental method for single-cell whole transcriptome sequencing of human bone tissue applicable to the present invention, exploration and verification were carried out from human bone tissue pretreatment to preparation of human bone tissue single-cell suspension and human bone cell single-cell whole transcriptome sequencing. The content of the present invention was finally obtained by consulting literature and multiple verifications. The method and results are shown in Comparative Examples 1 to 16 in Table 2 below. First, through Comparative Example 1, a mechanical crushing and grinding method was used to try to prepare a cell suspension of human bone tissue. The results are shown in Table 2. When Comparative Example 1 used a mechanical dispersion method to prepare a human bone single-cell suspension, only less than 100 intact cells were obtained, and the cell viability was less than 30%. After cell death, nucleic acid would degrade, and the number of cells obtained in the experiment was far from meeting the cell number requirement for high-throughput single-cell transcriptome sequencing. Therefore, a single-cell transcriptome experiment could not be performed. The enzymatic hydrolysis method is relatively mild and is superior to the mechanical dispersion method in maintaining cell integrity and controllability. Usually, the cell suspension obtained by the enzymatic hydrolysis method has the characteristics of few impurities and high viability. Therefore, comparative examples 2 to 4 used Miltenyi Biotec's Bone Marrow Dissociation Kit, MACSTissue Dissociation Kits, and Stemcell Technologies' Mouse Skeletal Tissue Dissociation Kit to prepare a human bone tissue cell suspension. The results showed that the cell viability was significantly increased, but the total amount of cells obtained was less than that obtained by mechanical dispersion. Human bone tissue is not easily broken, and its hardness and strength are relatively high, which greatly limits the efficiency of the enzyme entering the tissue to separate cells. Comparative examples 5 to 7 extended the working time of the enzymatic solution. The results showed that as the time increased, the number of cells obtained increased, but the total amount was still far from exceeding 10,000 cells. After overnight enzymatic hydrolysis, the cells died and broke due to too much stress. Comparative example 8 used a combined treatment method of mechanical dispersion and enzymatic hydrolysis, using slight mechanical dispersion to help the enzyme enter the tissue for cell separation. The results showed that the number of cells could reach 1,000, and the viability was qualified, but the yield was still far from that of single-cell transcriptome sequencing.

[0485] Table 2 Comparative Examples 1 to 8

[0486]

[0487]

[0488] By comparing the results of Example 8, two conclusions can be drawn: (1) The currently available kits for mouse bone and the commonly used enzymatic hydrolysis methods cannot be used to prepare single-cell suspensions of human hard bone tissue through simple optimization. (2) The hardness and strength of human hard bone tissue make it difficult to release cells, requiring the cells to be exposed before preparing a single-cell suspension. By consulting the structural characteristics of human bones and combining the components of human bones, the reason for the high hardness and high strength of bones is that hydroxyapatite accounts for 65% to 70% of the dry weight. Comparative Examples 9 to 14 try to use BL buffer (0.01M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02M TCEP, prepared with deionized water, pH = 5.5), BL buffer 2 (0.01M nitric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02M TCEP, prepared with deionized water, pH = 5.5), BL buffer 3 (0.01M perchloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02M TCEP, prepared with deionized water, pH = 5.5), BL buffer 4 (0.01 M hydroiodic acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5), BL buffer 5 (0.01 M hydrobromic acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP, prepared with deionized water, pH = 5.5), BL buffer 6 (0.005 M sulfuric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, 0.02 M TCEP (prepared with deionized water, pH = 5.5) was used to treat human hard bone tissue to reduce the hardness of the bone. The effectiveness of this step was evaluated by weighing the treated tissue. The results of Comparative Examples 9 to 14 showed that the effect after treatment with BL buffer was significant, the tissue hardness was significantly reduced, the hard bone surface became porous, and the tissue size and weight were significantly reduced. The hardness and mass of the tissues treated with BL buffer 2, BL buffer 3, BL buffer 4, and BL buffer 5 changed little within 15 minutes. It can be clearly observed that its effect was not as effective as BL buffer 1. Dilute nitric acid has strong oxidizing properties, which may damage cell membrane proteins and then destroy cell structure. At the same time, this is also fatal to the integrity of nucleic acids. Therefore, the risk of choosing dilute nitric acid as a tissue pretreatment reagent is extremely high. Hydroiodic acid, hydrobromic acid, and perchloric acid are highly corrosive and pose safety risks during use. Therefore, hydroiodic acid, hydrobromic acid, and perchloric acid cannot be used to treat tissues.Turbidity appeared in the tissue after treatment with BL buffer 6. Dilute sulfuric acid has strong water absorption and may cause cell dehydration and death. Dehydration of nucleic acids also increases the risk of degradation.

[0489] Table 3 Comparative Examples 9 to 14 Results

[0490]

[0491]

[0492] Therefore, dilute hydrochloric acid was selected as the component of BL buffer in the present invention. Comparative Examples 15 to 18 investigated the effectiveness of Tween 20, glutathione, and TCEP in BL buffer. The cell viability was preliminarily observed using enzymatic hydrolysis of pretreated tissue to reflect the state of cells in the tissue after pretreatment. Comparative Example 15 showed that after BL buffer treatment, the tissue could release 1×10 5cells, and the cell viability was greater than 90%, indicating that the cells were not severely stimulated and died during the pretreatment process; the results of Comparative Example 16 showed that the cell viability in BL buffer 7 (0.01M hydrochloric acid, 0.2% (m / v) glutathione, 0.02M TCEP, prepared with deionized water, pH = 5.5) was significantly reduced, and did not meet the requirements of subsequent experiments. Low concentrations of Tween 20 can reduce the tension and shear force on the cell membrane surface during the experiment, thereby reducing damage and destruction of the cell membrane. This helps to protect the integrity and function of cell membrane proteins. The results of Comparative Example 17 showed that the cell viability in BL buffer 8 (0.01M hydrochloric acid, 0.2% (v / v) Tween 20, 0.02M TCEP, prepared with deionized water, pH = 5.5) was significantly reduced, and did not meet the requirements of subsequent experiments. Glutathione can stabilize the pH value and osmotic pressure of the solution. Compared with the commonly used inorganic salts that stabilize the pH value and osmotic pressure of the solution, it does not produce stress stimulation on the cells itself, which can reduce the probability of false negative and false positive detection results. The results of Comparative Example 18 show that the results of BL buffer 9 (0.01M hydrochloric acid, 0.2% (v / v) Tween 20, 0.2% (m / v) glutathione, prepared with deionized water, pH = 5.5) are significantly lower than those of Comparative Example 15. Cell fragmentation can be clearly observed under a microscope, and the requirements for continuing the experiment cannot be met. The presence of TCEP can protect biological molecules such as cell membrane proteins that are easily oxidized. Through the reduction reaction, TCEP can eliminate or reduce the damage caused by oxidants to these molecules to maintain their functional activity. Because it is necessary to maintain reducing properties in an acidic environment, common reducing agents such as DTT and mercaptoethanol cannot be used in this environment to protect the cell membrane structure and maintain cell activity. Therefore, the optimal components of BL buffer are dilute hydrochloric acid, low concentration Tween 20, glutathione, TCEP, prepared with deionized water, pH = 5.5.

[0493] Table 4 Comparative Examples 15 to 18 Results

[0494]

[0495]

[0496] Comparative Examples 19 to 20 verified the effectiveness of BL buffer 10 (0.002 M hydrochloric acid, 0.05% Tween 20, 0.05% glutathione, 0.005 M TCEP, prepared with deionized water) and BL buffer 11 (0.015 M hydrochloric acid, 0.3% (v / v) Tween 20, 0.3% (m / v) glutathione, 0.03 M TCEP, prepared with deionized water). The results of Comparative Example 19 showed that low concentrations of hydrochloric acid could not effectively complete the pretreatment of hard bone tissue, and the cell yield decreased significantly, failing to meet the minimum requirements of subsequent experiments. The results of Comparative Example 20 showed that the cell viability was significantly reduced, failing to meet the minimum requirements of subsequent experiments for samples. Excessive hydrochloric acid and a low pH environment can damage cell membranes. Tween-20 is a non-ionic detergent that protects cell membranes at low concentrations. However, at high concentrations, Tween-20 molecules can insert into the lipid bilayer, disrupting the cell membrane structure and affecting cell activity. Glutathione reacts with reactive oxidants such as oxygen free radicals, thereby participating in the scavenging of oxygen free radicals. However, at high concentrations, glutathione's reaction can be too intense, disrupting the intracellular redox balance and causing cytotoxicity. High concentrations of TCEP can reduce proteins or peptides containing disulfide bonds (SS) to their corresponding sulfhydryl groups (-SH). This can damage proteins on the cell membrane surface, altering the cell membrane structure and affecting cell activity. Therefore, the components in the BL buffer should be within the specified components for pretreatment of complete human bone tissue, namely, BL buffer: 0.005-0.01M hydrochloric acid, 0.1-0.2% Tween 20, 0.1-0.2% glutathione, 0.01M-0.02M TCEP, prepared with deionized water, pH = 5.5-6.

[0497] Table 5 Comparative Examples 19-20 Results

[0498]

[0499]

[0500] Comparative Examples 21-22 verified the optimal treatment time of BL buffer in treating approximately 150mg of human hard bone tissue. The results showed that when the treatment time was less than 10min, the human hard bone tissue was not thoroughly de-inorganized, and the extracellular matrix and cells were not fully exposed, resulting in insufficient number of cells obtained by enzymatic hydrolysis. When the treatment time was greater than 15min, the extracellular matrix was overexposed in a slightly acidic environment, and the cell viability began to drop below 90%, which would bring hidden risks to subsequent experiments. The results of Comparative Examples 1-22 showed that BL buffer must be used to treat human hard bone tissue for a limited time within a limited composition and a limited concentration to achieve the expected treatment effect. Any of these changes will lead to the failure of the experiment or bury risks for the follow-up.

[0501] Table 6 Comparative Examples 21-22 Results

[0502]

[0503] After pretreatment of human hard bone tissue, the extracellular matrix and structural proteins were exposed, the main component of which is collagen. Comparative Examples 15 to 22 showed that using 0.2% collagenase I could separate cells from the tissue. In Comparative Example 23, collagenase II, which is generally believed to have a better enzymatic effect on hard tissue, was selected for cell separation. The results showed that the cell suspension prepared with 0.2% (m / v) collagenase II had a lower viability than the cell suspension obtained by collagenase I enzymatic hydrolysis, and the number of cells in the collagenase II group also decreased. When the hard bone tissue is treated with BL buffer, the extracellular matrix and structural proteins are directly exposed to the enzymatic hydrolysis solution. Compared with collagenase I, collagenase II will give cells a stronger stimulation, which is not conducive to maintaining high cell activity and low stress. Therefore, when decomposing cells in hard bone tissue, collagenase II, which is generally believed to be the preferred choice, was not selected. Instead, collagenase I, which has better efficiency, was selected as the main component of the BE solution to decompose the extracellular matrix and structural proteins. About 15% of the cells in the resulting cell suspension were clumped. In Comparative Examples 24 to 27, the method for treating cell clumping was verified. In Comparative Example 24, 0.1% (m / v) bovine serum albumin was added to the enzymatic solution. The results showed that the addition of bovine serum albumin did not solve the problem of cell clumping, and the addition of bovine serum albumin led to a decrease in cell yield. The presence of bovine serum albumin affected the enzymatic activity of collagenase I. In Comparative Example 25, 75 U / mL DNase I was added to the enzymatic solution. The results showed that the clumping situation did not improve. Combined with the cell viability of more than 90%, it was shown that the clumping was not caused by nucleic acids released by cell rupture. In Comparative Example 26, the effect of 0.25% (m / v) pancreatic enzyme on cell clumping was verified. The results showed that after treatment with 0.25% (m / v) pancreatic enzyme at 37°C for 3 minutes, cell clumping was significantly reduced, but cell viability was significantly decreased. The results showed that clumping was caused by the failure to destroy the intercellular connections. Destroying the intercellular connections can solve the clumping problem. It is well known that pancreatic enzyme has strong enzymatic activity and is suitable for tissues with high hardness and strength. However, the results showed that after only 3 minutes of treatment, the cell clumping had not yet completely dispersed and the cell viability had already decreased significantly. Therefore, it could not be selected as an experimental method. The action site of papain is also the intercellular connection. It has a mild effect and is used for enzymatic hydrolysis of tissues with simple structure, softness and low strength. In Comparative Example 27, the effect of 0.25% (m / v) papain on cell agglomeration was verified. The results showed that 0.25% (m / v) papain and 0.25% (m / v) collagenase I can complete the preparation of cell suspension of hard bone tissue after BL buffer treatment. All indicators of the cell suspension meet the requirements for risk-free conduction of the next experiment.Papain and collagenase I have a common feature that they require calcium ions as a coenzyme to improve the efficiency of the enzyme. Therefore, HBSS was selected as the buffer solution for the BE solution. In Comparative Examples 28 to 29, the enzymatic hydrolysis effect of using calcium- and magnesium-free HBSS and DPBS as buffer solutions was verified. The results showed that after replacing the buffer solution, the total cell count decreased significantly within 15 minutes, reflecting a significant decrease in enzyme activity. Therefore, a combination of HBSS, collagenase I, and papain was selected in the BE solution for cell separation of human bone tissue after BL buffer treatment.

[0504] Table 7 Comparative Examples 23 to 29

[0505]

[0506] Comparative Examples 30 and 31 verified the concentrations of the components in the BE solution. The results of Comparative Example 30 showed that BE Solution 2 (0.1% (m / v) collagenase I, 0.1% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092)) significantly reduced enzymatic digestion efficiency, decreased cell yield, and increased clumping. Prolonged enzymatic digestion time prolonged protease stimulation of cells, increasing the risk of false-positive and false-negative results. Therefore, BE Solution 2 should not be used for cell digestion. Comparative Example 31 showed that BE Solution 3 (0.3% (m / v) collagenase I, 0.3% (m / v) papain dissolved in 1× HBSS (Gibco, 14025092)) reduced enzymatic cell yield and viability. Using too high a collagenase concentration can cause strong stress to cells, posing a risk to experimental authenticity. Furthermore, increasing the concentration does not increase efficacy and is a waste of reagents. Therefore, the BE solution needs to be prepared according to the specified formula: 0.2-0.25% (m / v) collagenase I and 0.2-0.25% (m / v) papain are dissolved in 1× HBSS (Gibco, 14025092).

[0507] Table 8 Comparative Examples 30-31

[0508]

[0509] Comparative Examples 32 and 33 verified the working time limit of the BE solution. The results of Comparative Example 32 showed that when the BE solution was working for 10 minutes, the tissue could not be fully enzymatically hydrolyzed, the number of cells was small, and cell clumping still existed, and the experimental performance showed a significant decline. The results of Comparative Example 33 showed that when the BE solution was working for 25 minutes, cell activity decreased, and there was a risk in continuing the experiment. In summary, the components of the BE solution should be within a limited range, and experiments should be conducted on limited tissues according to limited methods to obtain a qualified human bone tissue cell suspension.

[0510] Table 9 Comparative Examples 32-33

[0511]

[0512] The prepared single-cell suspension needs to be polyadenylated before it can be used to complete the single-cell whole transcriptome sequencing experiment using a commercial kit. Due to the selective permeability of the cell membrane, polyadenylation enzyme cannot pass through the cell membrane to the cytoplasm and nucleus to carry out the polyadenylation enzymatic reaction. Usually, fixing cells can change the permeability of the cell membrane while maintaining the structure and expression of nucleic acids. Comparative Examples 34 to 36 explored commonly used cell fixation methods and cell fixatives with better performance. The nucleic acid fixation efficiency of the cells after extraction was judged by the bands of agarose gel electrophoresis. The results showed that the nucleic acid preservation state of the cells after PFA fixation had obvious advantages compared with the other two, and the RNA was more complete. However, judging from the quality inspection results, RNA still shows degradation. After RNA degradation, the structure and integrity of the nucleic acid decrease, and the enzyme binding site will be lost due to structural changes, which will directly affect the results of subsequent enzymatic reactions. Therefore, it is necessary to avoid changes in nucleic acids during PFA treatment of tissues. When living cells are stimulated by PFA, the cells will dehydrate. At the same time, changes in the extracellular physiological environment and stimulation will also be accompanied by nucleic acid dehydration, which will cause the nucleic acid in the cell to dehydrate and become unstable. Pectin and agarose have high hydration capacity and can adsorb water molecules and form a hydration layer. This hydration layer can maintain a moist environment inside the cell and reduce the dehydration and degradation of nucleic acids, thereby protecting the integrity of nucleic acids and the integrity of enzyme binding sites. This conclusion is demonstrated in Comparative Examples 37 to 39. The results of Comparative Example 37 show that the addition of RNase inhibitor does not improve the quality of RNA, and the addition of RNase inhibitor does not improve the quality of RNA, indicating that the degradation of nucleic acids is not caused by RNase. The results of Comparative Example 38 show that the addition of pectin further maintains the quality of nucleic acids and achieves the ideal quality. Comparative Example 39 attempted to use agarose as a protective agent component. However, because agarose contains galactoagglutinose, it forms a colloidal complex with DNA or RNA, which can severely affect RNA extraction and purification, as well as polyadenylation enzyme binding to RNA in subsequent experiments. Therefore, agarose cannot be used as a protective agent component. Therefore, PFA and pectin were selected as protective agent components for pretreatment of human scleroblasts.

[0513] Table 10 Comparative Examples 34 to 39

[0514] serial number Cell fixative Cell fixation method Experimental results Comparative Example 34 3% paraformaldehyde Fix at 4°C for 10 min RIN: 6.8 Comparative Example 35 Methanol Fix at -20℃ for 20 minutes RIN: 5.8 Comparative Example 36 RNAlater Fix at 4°C for 10 min RIN: 6.0 Comparative Example 37 3% paraformaldehyde, 4 U / ml RNase inhibitor Fix at 4°C for 10 min RIN: 6.9 Comparative Example 38 3% paraformaldehyde, 0.02% pectin Fix at 4°C for 10 min RIN: 7.8 Comparative Example 39 3% paraformaldehyde, 0.02% agarose Fix at 4°C for 10 min RIN: 6.8

[0515] Comparative Examples 40 and 41 explored the appropriate concentration ranges for the components of human osteoblast protective agents. The results of Comparative Example 40 showed that, under the treatment of human osteoblast protective agent 2 (1% (v / v) PFA, 0.03% (m / v) pectin, ddH2O), the nucleic acid quality was substandard, with severe nucleic acid degradation occurring during the process. The results of Comparative Example 41 showed that, after treatment with human osteoblast protective agent 3 (4% (v / v) PFA, 0.3% (m / v) pectin, ddH2O), most cells were broken. Excessive PFA and pectin concentrations lead to organelle dissolution, membrane rupture, and coagulation of cellular proteins, resulting in damage and deformation of the cell structure. Therefore, human osteoblast protective agents must use defined components and treat human osteoblasts at defined concentrations to achieve the desired RNA structure protection.

[0516] Table 11 Comparative Examples 40-41

[0517]

[0518] In Comparative Examples 42 to 43, the appropriate treatment time for treating human sclerostin cells with human sclerostin cell protective agents was explored. The results of Comparative Example 42 showed that when the treatment time was less than 9 minutes, the cell protective agent did not completely penetrate into the interior of the cells, and the cell protective agent may cause an imbalance of solutes inside and outside the cells, leading to cell rupture. The results of Comparative Example 43 showed that when the treatment time was longer than 10 minutes, the cell protective agent was treated for a long time, and PFA and pectin caused changes in the cell membrane structure, resulting in cell structure damage. In summary, human sclerostin cell protective agents should be used to treat human sclerostin cells according to a defined formula, within a defined concentration, and according to a defined method to achieve the desired effect.

[0519] Table 12 Comparative Examples 42-43

[0520]

[0521] Comparative Examples 44-45 explored the use of BL buffer and BE solution to treat mouse femur and human skeletal muscle tissue. The results of Comparative Example 44 showed that after treatment with BL buffer, approximately 200 mg of mouse femoral tissue was essentially unretained, leaving only the fibrous structure within the tissue. The cells in the suspension were completely broken, failing to meet the minimum cell viability requirements for single-cell transcriptome sequencing experiments. Furthermore, human skeletal muscle tissue treated with BL buffer and BE solution had extremely low cell yields and poor cell viability, failing to meet the requirements of cell-based experiments. This means that BL buffer and BE solution must be used in the prescribed order and according to established procedures to produce a human bone cell suspension that meets the standards for single-cell experiments. This is not applicable to other species or tissue types.

[0522] Table 13 Comparative Examples 44-45

[0523]

[0524] Comparative Example 46 explored the tissue utility of a human osteocyte protective agent. The results showed that human PBMC samples treated with the human osteocyte protective agent exhibited severe cell fragmentation and nucleic acid degradation. Cells from different tissues exhibit significant differences in intracellular and membrane structure due to their physiological environments and functions, and their tolerance to the agent also varies widely. In summary, a human osteocyte protective agent can only achieve its beneficial effect on human osteocytes when applied to a specific composition, concentration, and method of use.

[0525] Table 14 Comparative Example 46

[0526] serial number Cell type Cell treatment method Experimental results Comparative Example 46 Human PBMC Human osteoblast protection reagent Most cells were broken, RIN=3.7

[0527] The present invention relates to a pretreatment liquid BL buffer (0.005-0.01M hydrochloric acid, 0.1%-0.2% (v / v) Tween 20 (Solarbio, T8220), 0.1%-0.2% (m / v) glutathione (Solarbio, G8180), 0.01M-0.02M TCEP (Thermo Fisher, T2556), prepared with deionized water, pH=5.5-6) ​​suitable for preparing a single cell suspension from human sclerotium, and a method for using the BL buffer. According to the method provided by the present invention, de-inorganization treatment of human sclerotium tissue can be completed, hard matrix in the sclerotium can be removed, and extracellular matrix can be exposed to the buffer. The method is simple, rapid, and low-cost. The present invention relates to a reagent BE solution (0.2% to 0.25% (m / v) collagenase I (Solarbio, C8140); 0.2% to 0.25% (m / v) papain (Sigma, P4762) dissolved in 1× HBSS (Gibco, 14025092)) suitable for preparing a single-cell suspension from human scleral bone tissue after treatment with BL buffer, and a method for using the same. According to the established method of use in the present invention, a single-cell suspension of human scleral bone tissue after treatment with BL buffer can be prepared. This method efficiently completes the isolation of human scleral bone tissue cells in 15 minutes, filling the technical gap of the inability to effectively and efficiently prepare a high-viability single-cell suspension from human scleral bone tissue. Finally, in order to complete the single-cell whole transcriptome experiment, the present invention also covers a human sclerotin cell protective agent (2-3% (v / v) PFA (Solarbio, P1110), 0.05-0.1% (m / v) pectin (sigma, P7536), ddH2O) and its use method. Treating human sclerotin cells according to the established method of the present invention can effectively protect the nucleic acid structure of human sclerotin cells and the integrity of intracellular nucleic acids to provide high-quality risk-free experimental samples for subsequent polyadenylation experiments and single-cell whole transcriptome sequencing experiments. The present invention conducted a series of comparative example experiments during the exploration. According to the results of comparative examples 1 to 46, the present invention relates to a human sclerotin tissue pretreatment liquid BL buffer and its use method, a cell separation reagent BE solution suitable for human sclerotin tissue after BL buffer treatment and its use method, and a human sclerotin cell protective agent suitable for human sclerotin cells and its use method.The ideal efficacy can be achieved by treating human sclerotin tissue and human sclerotin tissue cells in accordance with the component composition, method of use, and order of use specified in the present invention, namely, obtaining a high-quality, risk-free human sclerotin single cell suspension treated with a human sclerotin cell protective agent. The suspension can be used for polyadenylation treatment and for single-cell whole transcriptome sequencing experiments using 10×Genomics ChromiumNext GEMAutomated Single Cell 3'cDNAKit v3.1 SOP after polyadenylation treatment. That is, there is a one-to-one compatibility relationship between the BL buffer and its method of use, the BE solution and its method of use, the human sclerostin cell protective agent, the human sclerostin tissue, the cells, and the products obtained by using the BL buffer, the BE solution, and the human sclerostin cell protective agent according to the established method for use in polyadenylation treatment and the single-cell whole transcriptome sequencing experiment after polyadenylation. That is, the above-mentioned reagents must be used in accordance with defined components, defined concentrations, defined methods of use, and defined targets in order to complete the experiment step by step, and obtain the treated human sclerostin cell suspension for use in polyadenylation treatment and the single-cell whole transcriptome sequencing experiment after treatment.

[0528] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A method for obtaining human bone tissue cells suitable for full transcriptome sequencing, characterized in that: A cell suspension of human sclerotin tissue is prepared using BL buffer, BE solution, and human sclerotin cell protective agent; the steps include: Step I, tissue cleaning and pretreatment: placing human sclerosing bone tissue in a centrifuge tube, soaking it in DPBS, inverting the centrifuge tube to clean the human sclerosing bone tissue, and pretreating it with pre-chilled BL buffer; using the BL buffer to perform a sclerosing demineralization treatment, adding the human sclerosing bone tissue to the BL buffer to perform the demineralization treatment, removing the hard matrix in the sclerosing bone and exposing the extracellular matrix to the buffer; the demineralization treatment temperature is 4°C to 6°C, the treatment time is 10 minutes to 15 minutes, and horizontal shaking is performed for 30 seconds every 2-3 minutes; Step II, tissue processing and enzymatic hydrolysis: The pretreated human bone tissue and BL buffer were passed through a 100 μm cell sieve, and the tissue was rinsed 2-3 times with pre-chilled PBS; the human bone tissue on the cell sieve was transferred to a culture dish, BE solution was added, the tissue was cut into small pieces, and BE solution was added; the culture dish was placed in a 37°C constant temperature incubator and enzymatic hydrolysis was performed for 15 minutes, with the culture dish shaken every 5 minutes; after the enzymatic hydrolysis was completed, the enzymatic hydrolysis solution was transferred to a new centrifuge tube using a pipette; Step III, cell separation and collection: Filter the enzymatic solution through a 40 μm cell sieve, rinse the cell sieve with pre-chilled DPBS containing 0.1% m / v BSA; centrifuge the filtrate in a horizontal centrifuge to collect the cells; Step IV: Protection treatment of human bone tissue cells: Step a, adding a human bone cell protective agent to a cell pellet obtained by centrifuging a human bone tissue cell suspension, resuspending the cell pellet, and mixing the suspension; Step b: Place the centrifuge tube used to store cells in step a at 4-6°C for 8-10 minutes; Step c: Add pre-chilled DPBS to the centrifuge tube, mix thoroughly by inverting it 4-5 times, and centrifuge it in a horizontal centrifuge to enrich the cells; The composition of the BE solution is: 0.2% to 0.25% m / v collagenase I and 0.2% to 0.25% m / v papain dissolved in 1× HBSS; The BL buffer comprises: 0.005-0.01 M hydrochloric acid, 0.1%-0.2% v / v Tween 20, 0.1%-0.2% m / v glutathione, 0.01 M-0.02 M tris(2-carboxyethyl)phosphine, prepared with deionized water, pH = 5.5-6; The human scleroblast protective agent comprises the following components: 2-3% v / v paraformaldehyde PFA, 0.05-0.1% m / v pectin, and ddH2O.

2. The method according to claim 1, wherein The method further comprises polyadenylation of human sclerotin cells: The polyadenylation of human sclerotin cells comprises the following steps: washing the human sclerotin cells obtained in step IV of claim 1 with a washing buffer and centrifuging the cell pellet using a polyadenylate polymerase mixture and mixing, and incubating the suspension to complete the polyadenylation of the human sclerotin cells; after the reaction is completed, centrifuging at 500×g and 4°C for 10 minutes to enrich the human sclerotin cells, and removing the enzyme mixture; the incubation condition is 37°C for 20 minutes; The washing buffer comprises: 1× polyadenylate polymerase reaction buffer, 0.8 U / μL protective RNase inhibitor, and ddH2O; The components of the poly(A) polymerase mixture are: 1× poly(A) polymerase reaction buffer, 24 U / μL poly(A) polymerase, 0.5 mM adenosine triphosphate (ATP), and 1.5 U / μL protective RNase inhibitor.

3. A method for whole transcriptome sequencing of human bone tissue cells not for the purpose of disease diagnosis, characterized in that: The method comprises the following steps: Step 1: preparing a human bone tissue cell suspension by the method according to claim 1 or 2, and enriching the human bone tissue cells to be sequenced by centrifugation; Step 2: washing and resuspending the human bone tissue cells obtained in step 1 with a DPBS solution containing bovine serum albumin, wherein the content of the bovine serum albumin is 0.1-0.2% m / v; Step 3: Calculate and adjust the concentration of human bone tissue cells in the resuspended solution in step 2; the concentration of human bone tissue cells is adjusted using a DPBS solution containing 0.1-0.2% m / v bovine serum albumin, and the adjusted concentration is 1×10 6 / mL; Step 4: Perform single-cell encapsulation and molecular experiments according to the standard operating procedures of the 3' single-cell transcriptome sequencing kit; Step 5: Quality check the library results and perform single-cell whole transcriptome sequencing and analysis.

4. The method according to claim 3, wherein In step 1, the human sclerosing bone cells are cells in a human sclerosing bone cell suspension obtained by treating with BL buffer and BE solution, washing with washing buffer, and finally treating with a polyadenylation enzyme mixture; The centrifugation conditions are 300-500×g, 4-6° C., and 8-10 min; and the washing times are 2-3 times.

5. The method according to claim 3, wherein In step 3, the concentration of human sclerotin cells is calculated using acridine orange staining, and the final concentration is 10 μg / mL.

6. A solution suitable for single-cell whole transcriptome sequencing of human osteocytes, characterized in that: The solution includes BL buffer, BE solution, and human scleroblast protective agent; The composition of BL buffer is: 0.005-0.01 M hydrochloric acid, 0.1%-0.2% v / v Tween 20, 0.1%-0.2% m / v glutathione, 0.01 M-0.02 M tris(2-carboxyethyl)phosphine, prepared with deionized water, pH = 5.5-6; The composition of BE solution is: 0.2% to 0.25% m / v collagenase I and 0.2% to 0.25% m / v papain dissolved in 1× HBSS; The components of the human osteoblast protective agent are: 2-3% v / v paraformaldehyde PFA, 0.05-0.1% m / v pectin, and ddH2O.

7. Use of the method according to any one of claims 3 to 5 in the culture of human sclerotinoma cells not for the purpose of disease diagnosis, the preparation of human sclerotinoma single cell suspension suitable for single cell transcriptome sequencing / single cell whole transcriptome sequencing, and the study of human sclerotinoma cell division.

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