Universal kit and method for preparing plant single cell nuclear suspension

Abstract

The invention provides a universal kit and method for preparing a plant single cell nuclear suspension. The kit comprises a cell nucleus purification system for performing density gradient centrifugation on a cell nucleus resuspension; the cell nucleus purification system comprises a Percoll gradient separation liquid, a cell nucleus purification liquid and a sucrose solution with the concentration of 2-2.5 mol / L, wherein the Percoll gradient separation liquid and the cell nucleus purification liquid are 10%-30% (v / v). The method for preparing the plant tissue mononuclear suspension is stable in effect, simple to implement and high in universality. The technology can be suitable for cell nucleus extraction of various plant species and various plant tissues, the completeness of the extracted cell nucleus is high, and the technology can be applied to plant single cell nucleus transcriptome sequencing.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to a kit and a method for preparing a single cell nuclear suspension, and more specifically relates to a universal kit and a method for preparing a plant single cell nuclear suspension. Background Art

[0002] Single-cell transcriptome sequencing technology for mammals has become increasingly mature and has promoted research progress in the field of animal cells, but the application of this technology in plant tissues is still limited. Unlike animal cells, plant cells contain cell walls, complex organelles, and secondary metabolites, so the preparation process of plant single cells is very complicated. The commonly used protoplast preparation method has many limitations. Plant cells of different tissue types have cell walls of different thicknesses and osmotic pressures, and plant cells of different species have cell walls and secondary metabolites with different components. Therefore, the protoplast preparation process is complicated and has poor universality. In addition, plant tissues with a high degree of lignification or frozen storage are not suitable for the preparation of protoplasts. At the same time, the protoplast preparation process will cause the expression of some stress-related genes, which will interfere with the results.

[0003] Compared with plant protoplasts, plant cell nuclei are more stable, smaller in size, and can be extracted from a variety of tissues, with a wider range of applications. At present, there are two main technologies for preparing cell nuclei for plant tissues. One is flow cytometry screening, which relies on large-scale instruments and equipment, requires a starting cell amount of millions, and the sorting process will cause secondary damage to the cells. The second is to obtain cell nuclei by density gradient centrifugation purification, and use Percoll (Percoll) low-density gradient separation liquid to separate cell nuclei from organelles, cell fragments, etc. This method has low operating requirements and strong feasibility. However, during the application of the density gradient centrifugation method, due to the different volumes and masses of cell nuclei, the Percoll ratio needs to be adjusted according to the situation of the cell nucleus, and the purification and separation effect is not stable enough due to centrifugal force fluctuations (such as excessive centrifugation). The cell nucleus is easy to precipitate at the bottom of the centrifuge tube and cannot be separated from the cell fragments. It is difficult to recycle and use, resulting in experimental failure. Chinese patent CN118956859A discloses a method for preparing a plant single cell nuclear suspension, specifically using an iodixanol solution to perform density gradient centrifugation on a nuclear extract to obtain a plant single cell nuclear suspension. However, this method still has problems of poor stability and poor universality during the density gradient centrifugation process. Summary of the invention

[0004] In order to solve the problems of poor universality and poor stability existing in the process of preparing plant single-cell nuclear suspensions in the prior art, the present invention provides a kit and method for preparing plant tissue single-cell nuclear suspensions with stable effects, simple implementation, and strong universality. This technology can be applied to the nuclear extraction of a variety of plant species and various plant tissues, and the extracted nuclei have high integrity and can be applied to plant single-cell nuclear transcriptome sequencing.

[0005] The technical solution adopted by the present invention is as follows: A universal kit for preparing plant single-cell nuclear suspensions, the kit includes a nuclear purification system for density gradient centrifugation of the nuclear resuspension; the nuclear purification system includes: a nuclear purification solution, a sucrose solution; the nuclear purification solution includes a 10% - 30% (v / v) Percoll gradient separation solution; the sucrose concentration in the sucrose solution is 2 - 2.5 mol / L, more preferably 2.3 mol / L; the conditions for density gradient centrifugation are centrifugation at 1000 - 1500×g for 5 - 15 min.

[0006] The present invention provides a plant tissue single nucleus extraction technique based on density gradient centrifugation. This technique combines a Percoll low-density gradient separation solution with a high-concentration sucrose solution. By adjusting the appropriate concentration ratio of the two, a nucleus purification system with two layers of liquid is formed. The upper layer is a 10%-30% (v / v) Percoll gradient separation solution, and the lower layer is a 2-2.5 mol / L sucrose solution. In the selection of the concentration of the Percoll gradient separation solution, the higher the concentration, the higher the density of the Percoll gradient separation solution. During the nucleus purification process, a Percoll gradient separation solution with too high a concentration will make it difficult for small nuclei to be centrifuged into the sucrose layer, such as rice nuclei; while a Percoll gradient separation solution with too low a concentration will cause large nuclei to easily enter the sucrose layer and precipitate to the bottom due to its lower density, resulting in the failure of nucleus purification. In order to reduce the experimental complexity caused by nuclear differences and improve the stability of the experiment, under the density gradient centrifugation conditions of centrifuging at 1000-1500×g for 5-15 min, the concentration of the Percoll gradient separation solution is recommended to be between 10% and 30%. The selection of the sucrose concentration mainly lies in the fact that a high-concentration sucrose has a higher density, which is beneficial to "supporting" the nuclei and at the same time makes large clump impurities sink to the bottom. After repeated tests by the inventor's team, a 2-2.5 mol / L sucrose solution not only has relatively simple preparation and storage, can be prepared in advance and directly used without the need to prepare it immediately before use or specifically leave it overnight, but also can ensure that the nuclei do not sink to the bottom of the tube under the density gradient centrifugation conditions of centrifuging at 1000-1500×g for 5-15 min and in combination with a 10%-30% (v / v) Percoll gradient separation solution concentration. When in use, the nucleus resuspension solution is added to the top layer of the nucleus purification system to form a density gradient centrifugation purification system with three layers of liquid. The upper layer is the nucleus resuspension solution, the middle layer is a 10%-30% (v / v) Percoll gradient separation solution, and the lower layer is a 2-2.5 mol / L sucrose solution. After density gradient centrifugation at 1000-1500×g for 5-15 min, this scheme can enrich the nuclei at the interface of the middle and lower layers, and there is still a distance of more than 0.5 mL of liquid surface from the bottom impurities, thus avoiding the precipitation of large-volume or high-quality nuclei to the bottom of the centrifuge tube. Therefore, this scheme has high stability. In addition, since the collected nuclei are in the middle of the Percoll layer and the sucrose layer and close to the sucrose layer, even if there is a large difference in the volume or mass of the nuclei in a certain plant tissue, the nuclei with larger mass or volume can still be stably distributed in the middle of the Percoll layer and the sucrose layer or at the top of the sucrose layer and will not precipitate at the bottom of the centrifuge tube, which is also convenient for collecting the nuclei, and there will be no large amount of Percoll gradient separation solution and sucrose solution mixed in the collected liquid, and there is no need for multiple washes later.However, the common prior art uses different concentrations of percoll layers for purification and centrifugation, and the nuclei often appear at the bottom of the centrifuge tube, resulting in failure of nucleus purification. Therefore, this solution has wide universality. In summary, the technical solution provided by the present invention has strong stability and wide universality. The nuclei are not easy to precipitate to the bottom of the tube. It can be applied to the extraction of nuclei from various plant species and various plant tissues. The extracted nuclei have high integrity and can be applied to plant single-cell nuclear transcriptome sequencing.

[0007] Preferably, the kit further comprises at least one of a tissue dissociation fluid and a cell nucleus washing fluid.

[0008] Preferably, the kit comprises: tissue dissociation fluid, cell washing fluid, cell nucleus purification system, cell nucleus washing fluid; the cell nucleus purification system comprises: cell nucleus purification fluid, sucrose solution; the cell nucleus purification fluid comprises: 10%~30% (v / v) Percoll gradient separation fluid; the sucrose concentration in the sucrose solution is 2~2.5mol / L.

[0009] Preferably, the tissue dissociation solution comprises: 600-1000 mM sucrose, 8-12 mM magnesium chloride, 20-30 mM Tris-HCl, 1-3 mM EGTA, 0.1-0.2 mM DTT, 0.1-0.2 mM PMSF, 0.3-0.5 U / μL RNase inhibitor. Wherein, DTT, PMSF, and RNase inhibitor are added before use. More preferably, the tissue dissociation solution comprises: 800 mM sucrose, 10 mM magnesium chloride, 25 mM Tris-HCl, 2 mM EGTA, 0.1 mM DTT, 0.1 mM PMSF, and 0.4 U / μL RNase inhibitor.

[0010] Preferably, the cell washing solution comprises: 300-500 mM sucrose, 8-12 mM magnesium chloride (MgCl 2)、20~30 mM tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl, pH=8.0), 1~3 mM ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA, pH=7.4), 1%~1.5% (w / v) Triton X-100 (Triton X-100), 0.1~0.2mM dithiothreitol (DTT), 0.1~0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.03~0.05 U / μL RNase inhibitor. Wherein, DTT, PMSF, and RNase inhibitor are added before use. More preferably, the cell washing solution comprises: 400 mM sucrose, 10mM magnesium chloride, 25 mM Tris-HCl, 2 mM EGTA, 1% (w / v) Triton X-100, 0.1 mM DTT, 0.1 mM PMSF, and 0.04 U / μL RNase inhibitor.

[0011] Preferably, the cell nucleus purification solution comprises: 10%-30% (v / v) Percoll gradient separation solution, 70%-90% (v / v) cell washing solution, and 0.03-0.05 U / μL RNase inhibitor, wherein the RNase inhibitor is added before use.

[0012] Preferably, the cell nucleus washing solution comprises: 300-500 mM sucrose, 8-12 mM magnesium chloride, 20-30 mMTris-HCl, 1%-1.5% (w / v) Triton X-100, 0.1-0.2 mM PMSF, 0.03-0.05 U / μL RNase inhibitor. Among them, PMSF and RNase inhibitor are added before use. More preferably, the cell nucleus washing solution comprises: 400 mM sucrose, 10 mM magnesium chloride, 25 mM Tris-HCl, 1% (w / v) Triton X-100, 0.1 mM PMSF, 0.04 U / μL RNase inhibitor.

[0013] In order to improve the extraction effect of plant single cell nuclei, the present invention also provides a kit including the above-mentioned tissue dissociation solution, cell cleaning solution, cell nucleus purification system, and cell nucleus cleaning solution. The technical route of the preparation method of plant single cell nucleus suspension provided by prior art CN118956859A still needs to use enzymatic solution to enzymolyze plant tissue to obtain plant protoplasts. Therefore, the universality of this method is not high, and the occurrence of stress response in the protoplast preparation process cannot be avoided. Different from the prior art, the kit provided by the present invention does not need to prepare protoplasts in the process of preparing single cell nucleus suspension, but can directly extract cell nuclei from plant tissues, and then purify the obtained cell nuclei. In order to obtain a stable high-quality plant single cell nucleus suspension and reduce or avoid cell nucleus damage, the present invention has been targeted to adjust other solutions in the kit according to the above-mentioned cell nucleus purification system. Specifically, the tissue dissociation solution, cell cleaning solution, cell nucleus purification system, and cell nucleus cleaning solution provided by the present invention are respectively added with different concentrations of sucrose, and the addition of sucrose can not only adjust the osmotic pressure of the solution and maintain the viscosity of the liquid, but also help to disperse chloroplasts and prevent nuclear adhesion. In the process of preparing plant single cell nuclear suspension, the plant sample is first dissociated using tissue dissociation solution. The purpose of tissue dissociation is to break the cell wall by physical methods. In order not to destroy the nucleus, it is necessary to make the cell plasmolysis as much as possible, which requires high concentrations of sucrose to increase the osmotic pressure, but too high osmotic pressure can also cause nuclear shrinkage. Therefore, in the present invention, the tissue dissociation solution includes 600-1000 mM sucrose, more preferably 800 mM sucrose, for better achieving plasmolysis of plant cells. In the subsequent cell washing process, it is necessary to reduce the concentration of sucrose in the cell washing solution, so as to disperse the organelles as much as possible while maintaining the nuclear shape, which is conducive to the processing of subsequent steps and reduces nuclear adhesion. After using a cell nucleus purification system with a high sucrose concentration for density gradient centrifugation purification, the collected fluid containing the cell nuclei contains a high concentration of sucrose. When added to the cell nucleus washing fluid with a low sucrose concentration, the overall sucrose concentration increases, thereby effectively avoiding premature precipitation of the cell nuclei and causing nuclear aggregation during the centrifugation of the cell nucleus washing fluid. At the same time, it can also reduce the sedimentation rate of impurities and improve the quality of the single cell nucleus suspension.

[0014] The present invention also provides a method for preparing a plant single cell nucleus suspension using the kit, the method comprising: performing density gradient centrifugation on the cell nucleus resuspension using a cell nucleus purification system.

[0015] Preferably, the method comprises the following steps: S1. Dissociate the plant samples using tissue dissociation solution, remove impurities, centrifuge, and collect the precipitate; S2. Resuspend the precipitate using a cell washing solution to obtain a cell nucleus resuspension solution; S3. Use a nucleus purification system to perform density gradient centrifugation on the nucleus resuspension, and collect the collection liquid containing nuclei; S4. Wash the collection liquid with a nucleus washing solution, resuspend the precipitate after centrifugation, and obtain a single nucleus suspension.

[0016] Preferably, step S1 includes: cutting the plant sample in a tissue dissociation solution, collecting the liquid and filtering it, and collecting the filtrate; centrifuging the filtrate at 0-4 °C and 20-40×g for 3-7 min, and collecting the supernatant; centrifuging the supernatant at 0-4 °C and 500-1000×g for 5-10 min, and collecting the precipitate. Filtration is preferably carried out using a filter membrane, and the pore size of the filter membrane is preferably 30-50 μm.

[0017] Preferably, the method of performing density gradient centrifugation on the nucleus resuspension using the nucleus purification system includes: taking the nucleus purification solution in a centrifuge tube, adding a sucrose solution to the bottom of the nucleus purification solution, adding the nucleus resuspension to the top of the nucleus purification solution, and centrifuging at 0-4 °C and 1000-1500×g for 5-15 min to form upper, middle, and lower layers of liquid; collecting the collection liquid at the interface between the middle and lower layers.

[0018] Preferably, step S4 includes: adding the nucleus washing solution to the collection liquid, centrifuging at 0-4 °C and 600-800×g for 3-5 min, collecting the bottom liquid and the precipitate, adding a PBS buffer solution containing bovine serum albumin, mixing evenly, centrifuging at 0-4 °C and 600-800×g for 3-5 min, and collecting the precipitate; resuspending the precipitate with a PBS buffer solution to obtain a single nucleus suspension. In step S4, generally, the nucleus washing solution three times the volume of the collection liquid is used to wash the collection liquid for the first time, and after centrifugation, the precipitate is washed a second time with a PBS buffer solution containing bovine serum albumin. Without multiple washings, a single nucleus suspension with high nucleus integrity and uniformity can be obtained, meeting the single cell sequencing loading conditions. Among them, adding bovine serum albumin is to protect the nucleus, maintain the viscosity of the liquid, and reduce the damage to the nucleus caused by pipetting.

[0019] The present invention also provides a single nucleus sample obtained by using the kit or by using the method described above.

[0020] The present invention also provides the application of the kit or the method or the single nucleus sample described above in plant single nucleus transcriptome sequencing.

[0021] The beneficial effects of the present invention: 1. The present invention constructs a three-layer density gradient system (nucleus resuspension solution - Percoll layer - sucrose layer). During centrifugation, the nuclei are enriched at the interface between the Percoll layer and the sucrose layer, and maintain a safety distance of more than 0.5 mL from the bottom of the centrifuge tube. This design effectively avoids the problem that nuclei are prone to sink to the bottom in the traditional single Percoll system and has better stability. The high-density characteristic of the sucrose layer can accommodate nuclei of different volumes or masses. Even in the presence of large heterogeneity, nuclei with larger mass or volume can still be stably distributed in the sucrose layer without precipitating at the bottom of the centrifuge tube. Moreover, this method does not require the preparation of protoplasts and can directly extract nuclei from different tissues of different plants to prepare a single-nucleus suspension, with better universality.

[0022] 2. The Percoll layer reduces nuclear membrane damage through a non-ionic coating, and the sucrose layer maintains the stability of nuclear structure through osmotic pressure. Under the synergistic action of the double-layer density medium and combined with the physical separation of impurities by gradient centrifugation, nuclei of different plant tissues can be stably obtained without relying on a flow cytometer, reducing debris contamination. The extracted nuclei have high integrity and uniformity, meeting the requirements of single-nucleus transcriptome sequencing for nuclear membrane integrity and RNA protection.

[0023] 3. The method provided by the present invention avoids the high cost of flow sorting and the stress response of protoplast preparation, becoming a general solution without enzymatic digestion, reducing the technical cost and difficulty, and shortening the operation time. Description of the Drawings

[0024] Figure 1 Results of density gradient centrifugation of nuclei extracted from rice leaves in Example 1 and Comparative Example 1 of the present invention.

[0025] Figure 2 Nuclei extracted from cotton leaves in Example 2 of the present invention.

[0026] Figure 3 Nuclei extracted from maize leaves in Example 3 of the present invention.

[0027] Figure 4 Nuclei extracted from maize root tips in Example 4 of the present invention.

[0028] Figure 5 Nuclei extracted from Arabidopsis thaliana leaves in Example 5 of the present invention. Detailed Embodiments

[0029] The following describes the embodiments of the present invention through specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0030] Unless otherwise specified, the methods used in the examples of the present invention are all conventional methods, and the reagents used are all commercially available.

[0031] The embodiment of the present invention provides a universal kit for preparing a plant single cell nucleus suspension, the kit comprising: tissue dissociation solution A, cell washing solution B, cell nucleus purification solution C, sucrose solution, and cell nucleus washing solution D. The cell nucleus purification solution C and the sucrose solution are used to form a cell nucleus purification system.

[0032] Prepare tissue dissociation solution A, cell washing solution B, cell nucleus purification solution C, and cell nucleus washing solution D according to the following ingredients.

[0033] Meanwhile, prepare 2.3 M sucrose solution and pre-cool it on ice.

[0034] The tissue dissociation solution A includes: 800 mM sucrose, 10 mM magnesium chloride, 25 mM Tris-HCl, 2 mM EGTA, 0.1 mM DTT, 0.1 mM PMSF, and 0.4 U / μL RNase inhibitor. Among them, DTT, PMSF, and RNase inhibitor are added before use and pre-cooled on ice.

[0035] The cell washing solution B includes: 400 mM sucrose, 10 mM magnesium chloride, 25 mM Tris-HCl, 2 mM EGTA, 1% (w / v) Triton X-100, 0.1 mM DTT, 0.1 mM PMSF, and 0.04 U / μL RNase inhibitor. Among them, DTT, PMSF, and RNase inhibitor are added before use and pre-cooled on ice.

[0036] The cell nucleus purification solution C includes cell nucleus purification solution C1, cell nucleus purification solution C2, and cell nucleus purification solution C3.

[0037] The cell nucleus purification solution C1 includes: 10% (v / v) Percoll gradient separation solution, 90% (v / v) cell washing solution B, and 0.04 U / μL RNase inhibitor. The RNase inhibitor is added before use and pre-cooled on ice.

[0038] The cell nucleus purification solution C2 includes: 20% (v / v) Percoll gradient separation solution, 80% (v / v) cell washing solution B, and 0.04 U / μL RNase inhibitor. The RNase inhibitor is added before use and pre-cooled on ice.

[0039] The cell nucleus purification solution C3 includes: 30% (v / v) Percoll gradient separation solution, 70% (v / v) cell washing solution B, and 0.04 U / μL RNase inhibitor. The RNase inhibitor is added before use and pre-cooled on ice.

[0040] The cell nucleus washing solution D includes: 400 mM sucrose, 10 mM magnesium chloride, 25 mM Tris-HCl, 1% (w / v) Triton X-100, 0.1 mM PMSF, and 0.04 U / μL RNase inhibitor. Among them, PMSF and RNase inhibitor are added before use and pre-cooled on ice.

[0041] Example 1 Extraction of cell nuclei from rice leaves Step S1-1. Operating on ice, tissue lysis: Take 1200 mg of rice leaves, place them in a 100 mm culture dish, add 3 mL of tissue dissociation solution A, and chop the tissue into 1 mm pieces with a blade. 3 Cut the cells and nuclei into small pieces, tilting the dish slightly so that the released cells and nuclei can flow with the liquid. Step S1-2. Operate on ice and collect cell nuclei: add 5 mL of tissue dissociation solution A to the above culture dish and cut for 2 minutes; filter the liquid through a 40 μm filter membrane into a pre-cooled 50 mL centrifuge tube, add 5 mL of tissue dissociation solution A to the remaining tissue and continue cutting for 2 minutes, transfer the liquid into the above 50 mL centrifuge tube in the same way, and finally use 2 mL of tissue dissociation solution A to transfer all tissues to the above 40 μm filter membrane and collect the liquid; Step S1-3. Remove impurities: Centrifuge at 4°C and 30×g for 5 min, discard the precipitate, and collect the supernatant; Step S1-4. Enriching cell nuclei: Centrifuge the supernatant from step S1-3 at 4°C and 800×g for 8 min, discard the supernatant, and collect the precipitate; Step S2. Operate on ice to remove organelle contamination: resuspend the precipitate from step S1-4 in 4 mL of cell washing solution B, mix by pipetting with a pipette with the tip cut off, and place on ice for 5 minutes to obtain a cell nucleus resuspension solution; Step S3-1. Prepare the cell nucleus purification system I on ice: take 2 mL of 10% Percoll cell nucleus purification solution C1 at the bottom of a 15 mL centrifuge tube, slowly add 2 mL of 2.3 M sucrose at the bottom of the cell nucleus purification solution C1 to form a layer, prepare two tubes as replicates, and record them as I-1 and I-2 respectively; Step S3-2. Operate on ice, cell nuclei purification: Take 1 mL of the cell nuclei resuspension solution from step S2 and slowly add it to the purification system from step S3-1 to form three layers of liquid, with the cell nuclei resuspension solution on the upper layer; centrifuge at 4°C and 1500×g for 10 min. The results are as follows: Figure 1 As shown, 1.2 mL of the collection fluid containing the cell nuclei at the interface between the middle layer and the lower layer was collected; Step S4. Operate on ice and wash the cell nuclei: add 3.6 mL of cell nuclei washing solution D to the collected solution in step S3-2, and mix by inversion; centrifuge at 4°C and 700×g for 5 min, discard the supernatant, retain 0.5 mL of the liquid and precipitate at the bottom, add 2 mL of PBS solution containing 0.5% (w / v) bovine serum albumin, and mix by pipetting; centrifuge at 4°C and 700×g for 3 min, discard the supernatant, and resuspend the precipitate in PBS to obtain a single cell nucleus suspension.

[0042] Comparative Example 1 Extraction of cell nuclei from rice leaves The difference between Comparative Example 1 and Example 1 is that in step S3-2, a cell nucleus purification system mainly based on Percoll gradient separation solution is prepared, and the remaining steps are the same.

[0043] Step S3-2: Operate on ice, cell nucleus purification: Prepare cell nucleus purification system II: Take 2 mL of 10% Percoll cell nucleus purification solution C1 at the bottom of a 15 mL centrifuge tube, slowly add 2 mL of 75% Percoll cell nucleus purification solution C4 at the bottom of the cell nucleus purification solution C1 to form layers, prepare two tubes as replicates, and record them as II-1 and II-2 respectively; The cell nucleus purification solution C4 includes: 75% (v / v) Percoll gradient separation solution, 25% (v / v) cell washing solution B, and 0.04 U / μL RNase inhibitor. The RNase inhibitor was added before use and pre-cooled on ice.

[0044] After density gradient centrifugation, the results are as follows Figure 1 As shown. Figure 1It can be found that after density gradient centrifugation using the cell nucleus purification system I provided in Example 1 of the present invention, the separation effect is more stable, and there is no obvious precipitation at the bottom after centrifugation. The liquid at different positions from the bottom of the centrifuge tube is taken for cell nucleus staining observation using a fluorescence microscope, and it can be seen that the cell nuclei are enriched in the middle layer. However, after density gradient centrifugation using the cell nucleus purification system II provided in Comparative Example 1, the stratification is not obvious enough, there is obvious precipitation at the bottom of the tube, and there is a slight difference between the tubes II-1 and II-2, and the stability is poor. The liquid at different positions from the bottom of the centrifuge tube is taken for cell nucleus staining observation, and it can be seen that the cell nuclei are dispersed at the bottom and difficult to recover.

[0045] Example 2 Extraction of nuclei from cotton leaves Step S1-1. Operate on ice, tissue lysis: Take 300 mg of cotton leaves, place them in a 60 mm culture dish, add 0.5-1 mL of tissue dissociation solution A, and chop the tissue into 1 mm pieces with a blade. 3 Cut the cells and nuclei into small pieces, tilting the dish slightly so that the released cells and nuclei can flow with the liquid. Step S1-2. Operate on ice and collect cell nuclei: add 1 mL of tissue dissociation solution A to the above culture dish and cut for 2 minutes; filter the liquid through a 40 μm filter membrane into a pre-cooled 50 mL centrifuge tube, add 1 mL of tissue dissociation solution A to the remaining tissue and continue cutting for 2 minutes, transfer the liquid into the above 50 mL centrifuge tube in the same way, and finally use 1 mL of tissue dissociation solution A to transfer all tissues to the above 40 μm filter membrane and collect the liquid.

[0046] Step S1-3. Same as Example 1; Step S1-4. Same as in Example 1; Step S2. Operate on ice to remove organelle contamination: resuspend the precipitate from step S1-4 in 1 mL of cell washing solution B, mix by pipetting with a pipette with the tip cut off, and place on ice for 5 minutes to obtain a nuclear resuspension solution; Step S3-1. Prepare the cell nucleus purification system on ice: take 2 mL of 30% Percoll cell nucleus purification solution C3 at the bottom of a 15 mL centrifuge tube, and slowly add 2 mL of 2.3 M sucrose at the bottom of the cell nucleus purification solution C3 to form a layer; Step S3-2. Operate on ice, cell nuclei purification: Take 1 mL of the cell nuclei resuspension solution from step S2 and slowly add it to the purification system from step S3-1 to form three layers of liquid, with the cell nuclei resuspension solution on the upper layer; centrifuge at 4°C and 1500×g for 5 min. The results are as follows: Figure 1 As shown, 1.2 mL of the collection fluid containing the cell nuclei at the interface between the middle layer and the lower layer was collected; Step S4. Same as Example 1.

[0047] Take 10 μL of the single cell nucleus suspension in step S4, add 10 μL of PI working solution dye in a 1:1 ratio and mix well. Use the countstar automatic counting plate to count. The results are shown in Figure 2 .

[0048] Example 3 Extraction of nuclei from corn leaves Example 3: Using corn leaves as samples, extracting nuclei from them to prepare single nucleus suspension, the specific steps are the same as in Example 2. Take 10 μL of corn leaf single nucleus suspension, add 10 μL of PI working solution dye in a 1:1 ratio and mix well, use the countstar automatic counting plate to count, the results are shown in Figure 3 .

[0049] Example 4 Extraction of nuclei from corn root tips Example 4: Using corn root tips as samples, extracting nuclei from them to prepare single cell nucleus suspension, the specific steps are the same as in Example 2. Take 10 μL of corn root tip single cell nucleus suspension, add 10 μL of PI working solution dye in a 1:1 ratio and mix well, use the countstar automatic counting plate to count, the results are shown in Figure 4 .

[0050] Example 5 Extraction of nuclei from Arabidopsis leaves Example 5 Arabidopsis leaves were used as samples to extract nuclei and prepare single nucleus suspension. The specific steps were the same as those in Example 2, except that the 30% percoll nucleus purification solution C3 in step S3-1 was changed to 10% percoll nucleus purification solution C1. 10 μL of Arabidopsis leaf single nucleus suspension was taken, 10 μL of PI working solution dye was added in a 1:1 ratio, and the cells were counted using a countstar automatic counting plate. The results are shown in Figure 5 .

[0051] In summary of the above embodiments and comparative examples, the present invention constructs a three-layer density gradient system (nucleus resuspension-Percoll layer-sucrose layer). During the centrifugation process, the nuclei are enriched at the interface of the Percoll layer and the sucrose layer, and a safe distance of more than 0.5 mL is maintained from the bottom of the centrifuge tube. This can effectively avoid the problem of the nuclei easily sinking to the bottom in the traditional single Percoll system, and has better stability. The high-density characteristic of the sucrose layer can accommodate nuclei of different volumes or masses. Even if there is great heterogeneity, nuclei with larger masses or larger volumes can still be stably distributed in the sucrose layer without settling at the bottom of the centrifuge tube. Moreover, this method does not require the preparation of protoplasts, and can directly extract nuclei from different tissues of different plants to obtain a stable single nucleus suspension, avoiding debris contamination, and the extracted nuclei have high integrity and uniformity ( Figure 2-Figure 5), meeting the requirements of single-cell nuclear transcriptome sequencing for nuclear membrane integrity and RNA protection, and achieving the conditions for single-cell sequencing.

[0052] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A universal kit for preparing plant single cell nucleus suspension, characterized in that: The kit includes a cell nucleus purification system for performing density gradient centrifugation on a cell nucleus resuspension; The cell nucleus purification system comprises: cell nucleus purification solution and sucrose solution; The cell nucleus purification solution includes 10% to 30% (v / v) Percoll gradient separation solution; The sucrose concentration in the sucrose solution is 2-2.5 mol / L; The density gradient centrifugation condition is 1000-1500×g for 5-15 min.

2. The kit according to claim 1, characterized in that The cell nucleus purification solution includes: 10%-30% (v / v) Percoll gradient separation solution, 70%-90% (v / v) cell washing solution; The cell washing solution includes: 300~500 mM sucrose, 8~12 mM magnesium chloride, 20~30 mM Tris-HCl, 1~3 mM EGTA, 1%~1.5% (w / v) Triton X-100, 0.1~0.2 mM dithiothreitol, 0.1~0.2 mM phenylmethylsulfonyl fluoride, and 0.03~0.05 U / μL RNase inhibitor.

3. The kit according to claim 1, characterized in that The kit further comprises at least one of a tissue dissociation solution, a cell washing solution, and a cell nucleus washing solution; The tissue dissociation solution includes: 600-1000 mM sucrose, 8-12 mM magnesium chloride, 20-30 mM Tris-HCl, 1-3 mM EGTA, 0.1-0.2 mM dithiothreitol, 0.1-0.2 mM phenylmethylsulfonyl fluoride, and 0.3-0.5 U / μL RNase inhibitor; The cell washing solution includes: 300-500 mM sucrose, 8-12 mM magnesium chloride, 20-30 mM Tris-HCl, 1-3 mM EGTA, 1%-1.5% (w / v) Triton X-100, 0.1-0.2 mM dithiothreitol, 0.1-0.2 mM phenylmethylsulfonyl fluoride, and 0.03-0.05 U / μL RNase inhibitor; The cell nucleus washing solution includes: 300~500 mM sucrose, 8~12 mM magnesium chloride, 20~30 mM Tris-HCl, 1%~1.5% (w / v) Triton X-100, 0.1~0.2 mM phenylmethylsulfonyl fluoride, and 0.03~0.05 U / μL RNase inhibitor.

4. A method for preparing a plant single cell nucleus suspension using the kit according to any one of claims 1 to 3, characterized in that: The method comprises: using a cell nucleus purification system to perform density gradient centrifugation on a cell nucleus resuspension.

5. The method according to claim 4, characterized in that The method comprises the following steps: S1. Dissociate the plant samples using tissue dissociation solution, remove impurities, centrifuge, and collect the precipitate; S2. Resuspend the precipitate using a cell washing solution to obtain a cell nucleus resuspension solution; S3. performing density gradient centrifugation on the cell nucleus resuspension using a cell nucleus purification system to collect the collection fluid containing the cell nuclei; S4. Wash the collected fluid with cell nucleus washing solution, resuspend the precipitate after centrifugation to obtain a single cell nucleus suspension.

6. The method according to claim 5, characterized in that Step S1 includes: The plant samples were cut in tissue dissociation solution, the liquid was collected and filtered, and the filtrate was collected; The filtrate was centrifuged at 0-4°C and 20-40 × g for 3-7 min, and the supernatant was collected; The supernatant was centrifuged at 0-4°C and 600-1000×g for 5-10 min, and the precipitate was collected.

7. The method according to claim 4 or 5, characterized in that The method of using the cell nucleus purification system to perform density gradient centrifugation on the cell nucleus resuspension comprises: Take the cell nucleus purification solution into a centrifuge tube, add sucrose solution to the bottom of the cell nucleus purification solution, add the cell nucleus resuspension solution to the top of the cell nucleus purification solution, and centrifuge at 0-4°C and 1000-1500×g for 5-15 min to form three layers of liquid: upper, middle and lower; The collection liquid at the interface between the middle layer and the lower layer was collected.

8. The method according to claim 5, characterized in that Step S4 includes: Add cell nucleus washing solution to the collection solution, centrifuge at 0~4℃, 600~800×g for 3~5 min, collect the bottom liquid and precipitate, add PBS buffer containing bovine serum albumin, mix well, centrifuge at 0~4℃, 600~800×g for 3~5 min, and collect the precipitate; The pellet was resuspended in PBS buffer to obtain a single cell nucleus suspension.

9. A single cell nucleus sample obtained by processing using the kit according to any one of claims 1 to 3 or using the method according to claim 4.

10. Use of the kit according to any one of claims 1 to 3, the method according to claim 4, or the single cell nucleus sample according to claim 9 in plant single cell nucleus transcriptome sequencing.

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