Single cell nucleus extraction method suitable for single cell research of various plant species and various tissue types

Abstract

The invention relates to a plant tissue single cell nucleus preparation method, which comprises: (1) adding a cell nucleus extraction buffer solution to a plant tissue, and continuously crushing the plant tissue for 2-5 min to obtain a crushed sample; (2) collecting supernate from the sample, and collecting cell nucleuses from the supernate; (3) resuspending the cell nucleus by using a cell nucleus extraction buffer solution to prepare a cell nucleus suspension; (4) carrying out cell flow sorting on the cell nucleus suspension to obtain sorted cell nucleuses; wherein the cell nucleus extraction buffer solution comprises the following components with working concentrations: 1 * of NIB, 1 * of a protease inhibitor Cocktail, 0.4 U / [mu] L of an RNase inhibitor and 1 mM of DTT.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for extracting single cell nuclei suitable for single cell research of various plant species and various tissue types. BACKGROUND

[0002] In recent years, high-throughput single cell transcriptome research has rapidly developed in the field of animal and human research. Although relatively small-scale single cell characterization has been successfully performed using capillary-based methods in reproductive cells during maize development and leaf mesophyll cells in rice, there are fewer related studies in the field of plants, especially using high-throughput technologies such as 10x Genomics or Drop-seq, and most of them are characterization of protoplasts produced in Arabidopsis roots. One of the main reasons for the scarcity of research tissue types is that plant cells are naturally limited by cell walls, and the cell walls need to be removed to release protoplasts as single cells. This process has been fully tested in Arabidopsis roots, but it is still difficult in many other plant species or tissues.

[0003] Due to the differences in the toughness of the cell wall and the content of secondary metabolites in different plant species and plant tissues, it is extremely challenging to uniformly generate protoplasts from all cells. Moreover, enzyme digestion and subsequent cleaning processes during protoplast isolation can trigger stress responses and affect the transcriptome. In addition, traditional single cell sequencing methods that rely on protoplasts require a large amount of plant material. Therefore, there is an urgent need for a method that does not require protoplasts to expand the application of large-scale single cell analysis in plants.

[0004] Traditional techniques mainly rely on plant single cell transcriptome sequencing techniques that depend on protoplasts, which have the following defects:

[0005] 1. Traditional techniques require a large amount of plant tissue, consuming a large amount of sampling time and human and material resources.

[0006] 2. Due to the toughness of the cell wall and the complexity of secondary metabolites in different plant species and plant tissues, it is extremely challenging to uniformly extract protoplasts from different plant species and different tissues.

[0007] 3. The step of releasing protoplasts by enzymatic digestion of cell walls in traditional methods to some extent stresses plant protoplasts, causing cell damage or loss of viability, and affecting the gene expression of plant cells themselves. SUMMARY

[0008] Therefore, it is necessary to provide a method for preparing single cell nuclei suitable for multiple plant species and multiple tissues according to an embodiment of the present application.

[0009] The technical scheme comprises the following:

[0010] A method for preparing a single nucleus of a plant tissue, the method comprising the steps of:

[0011] (1) adding a nucleus extraction buffer to the plant tissue, continuously crushing the plant tissue, and preparing a crushed sample;

[0012] (2) collecting supernatant from the sample, and collecting nuclei from the supernatant;

[0013] (3) resuspending the nuclei with the nucleus extraction buffer, and preparing a nuclei suspension; and

[0014] (4) subjecting the nuclei suspension to cell flow sorting, and obtaining sorted single nuclei;

[0015] wherein the nucleus extraction buffer comprises the following components at the following working concentrations:

[0016] NIB 1x, protease inhibitor Cocktail 1x, RNase inhibitor 0.4 U / μL, and DTT 1 mM;

[0017] The plant tissue is derived from soybean, Leymus chinensis, and Sesbania cannabina.

[0018] In one embodiment, the plant tissue is continuously crushed for 2 min ~ 5 min.

[0019] In one embodiment, the plant tissue comprises roots, stems, leaves, and whole plants.

[0020] In one embodiment, the plant tissue comprises soybean stem tip meristem, Leymus chinensis seedling, Sesbania cannabina nodule, Sesbania cannabina root, Sesbania cannabina stem, Sesbania cannabina leaf, or Sesbania cannabina root tip.

[0021] In one embodiment, in step (2), the supernatant is subjected to a first filtration.

[0022] In one embodiment, in step (3), after resuspending the nuclei, the nuclei are subjected to a second filtration.

[0023] In one embodiment, the nuclei are collected by centrifugation at 300xg ~ 1000xg.

[0024] In one embodiment, the parameters for cell flow sorting comprise one or more of the following conditions:

[0025] (1) FSC channel value is 0.5% ~ 2.0%;

[0026] (2) sorting pressure is 1 ~ 3;

[0027] (3) FSC value is 3-5; and,

[0028] (4) BSC value is 27.0%-30.5%.

[0029] In one embodiment, the size of the cell strainer used in the first filtration is 40 μm; and / or, the size of the cell strainer used in the second filtration is 20 μm-30 μm.

[0030] In one embodiment, after step (1) is completed, it further includes observing the number and state of the nuclei in the sample. Optionally, when the number of nuclei in the sample is small, the precipitate after centrifugation of the sample is broken accordingly.

[0031] A single-nucleus transcriptome sequencing method, the method comprising the step of preparing single nuclei using the preparation method.

[0032] In one embodiment, it further includes the step of sequencing the single nuclei.

[0033] Compared with the traditional technology, the present application has the following beneficial effects:

[0034] The present application provides a single-nucleus extraction method independent of protoplasts, which overcomes the problems of large sample demand, complex operation, and severe cell damage in traditional technology. This method improves the efficiency and reliability of the operation, expands the application range of plant single-cell research, and has the following advantages:

[0035] 1. Low sample demand: only a small amount of plant tissue is needed to obtain a sufficient number of high-quality nuclei, saving labor and time costs.

[0036] 2. Strong universality: suitable for various plant species and tissue types.

[0037] 3. High nucleus integrity: effectively reduces nucleus adhesion and damage, ensuring sequencing data quality.

[0038] 4. Simple operation: simplifies the operation process, saving labor and time costs. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Comparison before and after optimization of single-nucleus isolation technology for soybean stem tip meristem;

[0040] Figure 2 Comparison before and after optimization of single-nucleus isolation technology for Leymus mollis seedlings;

[0041] Figure 3 Comparison before and after optimization of single-nucleus isolation technology for Sesbania cannabina root nodules;

[0042] Figure 4 Comparison of single cell nucleus isolation technique before and after optimization for roots of Sesbania cannabina;

[0043] Figure 5 Comparison of single cell nucleus isolation technique before and after optimization for stems of Sesbania cannabina;

[0044] Figure 6 Comparison of single cell nucleus isolation technique before and after optimization for leaves of Sesbania cannabina;

[0045] Figure 7 Comparison of single cell nucleus isolation technique before and after optimization for root tips of Sesbania cannabina. DETAILED DESCRIPTION

[0046] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced in a variety of ways beyond those specifically described herein, and that the present application is not limited to the specific embodiments disclosed below.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0048] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0049] English abbreviations referred to herein:

[0050] NIB (Nuclei isolation buffer): nuclei isolation buffer;

[0051] PBS (Phosphate-Buffered Saline): phosphate-buffered saline;

[0052] Chopping: (with a blade) chopping;

[0053] DAPI (4', 6-Diamidino-2-phenylindole): 4', 6-diamidino-2-phenylindole;

[0054] RNase inhibitor: ribonuclease inhibitor;

[0055] BSA (Bovine Serum Albumin): bovine serum albumin;

[0056] FSC (Forward Scatter): forward scattered light;

[0057] BSC (Back Scatter): back scattered light.

[0058] To solve the problems in the prior art, the application provides a single cell nucleus preparation method suitable for multiple plant species and multiple tissues. The single cell nucleus prepared by the method has high stability and integrity and is basically free of cell adhesion, and is suitable for high-throughput single cell transcriptome library construction of multiple plant species and tissues, and has wide applicability.

[0059] Based on this, one embodiment of the application provides a single cell nucleus preparation method for plant tissues, comprising the following steps:

[0060] (1) adding a cell nucleus extraction buffer to plant tissues, continuously crushing the plant tissues, and obtaining a crushed sample.

[0061] In one specific example, the plant tissue sources include soybean, Leymus chinensis and Sesbania cannabina. In one specific example, the plant tissues include roots, stems, leaves and whole plants. In one specific example, the plant tissues include soybean shoot apical meristems, Leymus chinensis seedlings, Sesbania cannabina root nodules, Sesbania cannabina roots, Sesbania cannabina stems, Sesbania cannabina leaves or Sesbania cannabina root tips.

[0062] In one specific example, the time for continuously crushing the plant tissues is 2 min ~ 5 min.

[0063] In one specific example, after step (1) is completed, it further comprises observing the number and state of the cell nuclei in the sample.

[0064] Optionally, when the number of cell nuclei in the sample is small, the precipitate after centrifugation of the sample is crushed, and this step is repeated until a sufficient amount of cell nuclei is obtained.

[0065] (2) centrifuging the sample to obtain supernatant, and collecting cell nuclei from the supernatant.

[0066] In one specific example, in step (2), the supernatant is filtered for the first time.

[0067] In one specific example, the size of the cell sieve for the first time filtering is 40 μm.

[0068] In one specific example, the cell nuclei are collected by centrifugation.

[0069] (3) resuspending the cell nuclei with a cell nucleus extraction buffer to prepare a cell nucleus suspension.

[0070] In one specific example, the nuclear extraction buffer includes the following working concentrations of components:

[0071] NIB 1x, Protease Inhibitor Cocktail 1x, RNase Inhibitor 0.4 U / µL, and DTT 1 mM.

[0072] In one specific example, in step (3), resuspending the nuclear pellet further includes a second filtration.

[0073] In one specific example, the second filtration uses a cell strainer with a size of 20 µm~30 µm.

[0074] (4) the nuclear suspension is subjected to cell flow sorting to obtain sorted nuclei.

[0075] Compared with the prior art, the method of the present application significantly reduces the sample requirement, simplifies the operation process, maintains the integrity of the transcript information, saves the labor and time cost, and greatly expands the application range of plant single-cell transcriptomics. Taking soybean stem tip meristem single-cell research as an example, compared with the traditional single-cell transcriptome sequencing method which requires at least 500 Arabidopsis thaliana apical meristems to produce enough protoplasts for library construction, the method of the present application only needs 50 apical meristems to obtain a sufficient number of high-quality nuclei for library construction and sequencing.

[0076] In addition, the system of the present application optimizes the single-cell nuclear isolation technology. By adjusting the composition and concentration of the nuclear extraction buffer, the tissue treatment time, the centrifugation parameters, the nuclear filtration parameters, and the flow sorting parameters, the stability and integrity of the nuclei are significantly improved, the adhesion and damage of the nuclei are effectively reduced, and the sequencing data quality is improved. The technology has been successfully extended to the single-cell research of soybean stem tip meristem, Leymus chinensis seedlings, and Sesbania cannabina root nodules, roots, stems, and leaf tissues. The optimization of each step is based on the special needs of specific species and tissue types, ensuring the adaptability of different plant species and tissues.

[0077] In one specific example, the centrifugation conditions include 300xg~1000xg centrifugation for 3 min~5 min.

[0078] In one specific example, the cell flow sorting parameters include one or more of the following conditions:

[0079] (1) the FSC channel value is 0.5%~2.0%;

[0080] (2) the sorting pressure is 1~3;

[0081] (3) the FSC value is 3~5;

[0082] (4) BSC value is 27.0%~30.5%.

[0083] In one specific example, the plant tissue is soybean shoot apical meristem, and the method for preparing single cell nuclei comprises one or more of the following conditions:

[0084] The plant tissue is crushed for 2 min~3 min;

[0085] The cell screen size for the first filtration is 40 μm;

[0086] The cell screen size for the second filtration is 20 μm;

[0087] The centrifugal rate is 1000×g;

[0088] The parameters for cell flow sorting include FSC channel value is 0.5%, sorting pressure is 2, FSC value is 4, and BSC value is 29.0%.

[0089] In one specific example, the plant tissue is Leymus mollis seedling, and the method for preparing single cell nuclei comprises one or more of the following conditions:

[0090] The plant tissue is crushed for 2 min~3 min;

[0091] The cell screen size for the first filtration is 40 μm;

[0092] The cell screen size for the second filtration is 30 μm;

[0093] The centrifugal rate is 300×g;

[0094] The parameters for cell flow sorting include FSC channel value is 2.0%, sorting pressure is 2, FSC value is 3, and BSC value is 27.0%.

[0095] In one specific example, the plant tissue is Sesbania cannabina nodule, and the method for preparing single cell nuclei comprises one or more of the following conditions:

[0096] The plant tissue is crushed for 5 min;

[0097] The cell screen size for the first filtration is 40 μm;

[0098] There is no second filtration;

[0099] The centrifugal rate is 500×g;

[0100] The parameters for cell flow sorting include FSC channel value is 1.0%, sorting pressure is 2, FSC value is 5, and BSC value is 30.5%.

[0101] In one specific example, the plant tissue is a root of Sesbania cannabina, and the method of preparing single nuclei of the plant tissue comprises one or more of the following conditions:

[0102] the plant tissue is crushed for 2 min ~ 3 min;

[0103] the first filtration cell strainer size is 40 μm;

[0104] there is no second filtration;

[0105] the centrifugation rate is 300 x g;

[0106] the parameters of cell flow sorting include FSC channel value of 2.0%, sorting pressure of 3, FSC value of 5, and BSC value of 28.0%.

[0107] In one specific example, the plant tissue is a stem of Sesbania cannabina, and the method of preparing single nuclei of the plant tissue comprises one or more of the following conditions:

[0108] the plant tissue is crushed for 5 min;

[0109] the first filtration cell strainer size is 40 μm;

[0110] there is no second filtration;

[0111] the centrifugation rate is 300 x g;

[0112] the parameters of cell flow sorting include FSC channel value of 2.0%, sorting pressure of 2, FSC value of 5, and BSC value of 28.0%.

[0113] In one specific example, the plant tissue is a leaf of Sesbania cannabina, and the method of preparing single nuclei of the plant tissue comprises one or more of the following conditions:

[0114] the plant tissue is crushed for 5 min;

[0115] the first filtration cell strainer size is 40 μm;

[0116] the second filtration cell strainer size is 30 μm;

[0117] the centrifugation rate is 300 x g;

[0118] the parameters of cell flow sorting include FSC channel value of 2.0%, sorting pressure of 1, FSC value of 5, and BSC value of 28.0%.

[0119] In one specific example, the plant tissue is a root tip of Sesbania cannabina, and the method of preparing single nuclei of the plant tissue comprises one or more of the following conditions:

[0120] the plant tissue is crushed for 2 min ~ 3 min;

[0121] The first filtration cell screen size is 40 μm;

[0122] No second filtration;

[0123] The centrifugation rate is 300 x g;

[0124] The parameters of cell flow sorting include FSC channel value of 2.0%, sorting pressure of 1, FSC value of 3, and BSC value of 28.5%.

[0125] An embodiment of the present application provides the single cell nucleus suspension prepared by the preparation method.

[0126] An embodiment of the present application provides a single cell nucleus transcriptome sequencing method, which comprises the step of preparing single cell nuclei by the preparation method.

[0127] In one specific example, the method further comprises the step of sequencing the single cell nuclei.

[0128] Embodiments of the present application will be described in detail below with reference to examples. It should be understood that the examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferably referred to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0129] In the following specific examples, the measurement parameters of raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed for the temperature and time parameters.

[0130] The single cell nucleus preparation method suitable for plant multi-species and multi-tissues in the following examples comprises:

[0131] All operations are performed on ice or at 4 degrees, and all operations need to be strictly prevented from RNase contamination.

[0132] 1. Place the glass dish after removing RNase on the metal module on ice, and suck 500 μl of prepared cell nucleus extraction buffer (1 x NIB) into the glass dish.

[0133] 2. Take at least 10 mg of plant tissue and place it in the glass dish, immerse the sample in 1 x NIB, and immediately chop the sample quickly with a blade to release the cell nuclei.

[0134] 3. After chopping the plant tissue for 2-5 min, pipette all the NIB solution from the dish into a pre-chilled 15 ml centrifuge tube. If the sample volume is large, you can add another 0.5-1 ml of lx NIB solution to rinse the chopped tissue into the centrifuge tube. Add DAPI (1 mg / ml) staining solution at a ratio of 1:1000 to stain the nuclei.

[0135] Important: The chopping time of plant tissue needs to be adjusted according to the plant species or tissue type.

[0136] 4. Take 1 μl of the nuclei solution onto a glass slide and examine under a microscope to observe the number and status of the nuclei.

[0137] 5. If the number of nuclei is too small, centrifuge the 100 μl of the nuclei solution in the 15 ml centrifuge tube at 4°C for 1 min, and collect the supernatant into a new 15 ml centrifuge tube. The nuclei are in the supernatant after centrifugation, and they can be filtered into a pre-chilled 50 ml centrifuge tube. The pellet can be used for further chopping to release the nuclei.

[0138] 6. Repeat steps 3-5 until enough nuclei are obtained. Transfer all the supernatant to a 40 μm cell strainer and filter into a pre-chilled 50 ml centrifuge tube. (The number of chopping times needs to be adjusted according to the number and status of the nuclei collected from different species and tissues. If the number of nuclei is not enough, further chopping is needed to release the nuclei. If the sample volume is large, the nuclei should not be chopped too much, which can cause the nuclei to be chopped too finely.)

[0139] 7. After three times of chopping and filtering through a 40 μm cell strainer, at least about 5 ml of nuclei solution can be collected. Take 1 μl of the nuclei solution for microscopic examination to determine whether the number of nuclei is sufficient.

[0140] 8. Transfer all the solution from the 50 ml centrifuge tube to a pre-chilled new 15 ml centrifuge tube, centrifuge at 1,000 g at 4°C for 5 min, and collect the nuclei.

[0141] Important: The centrifugation parameters need to be adjusted according to the plant species or tissue type.

[0142] 9. After centrifugation, pipette 1 μl of the supernatant for microscopic examination. If the number of nuclei in the supernatant is not large (normal case), you can discard the supernatant and collect the nuclei pellet. Important: Under normal circumstances, a nuclei pellet can be seen after centrifugation, but it is not possible to centrifuge all the nuclei in the supernatant into the pellet.

[0143] 10. Pipette 400 μl NIB (without DAPI) to resuspend the nuclei pellet, place in a 20 μm cell strainer, filter into a pre-chilled 1.5 ml centrifuge tube. Gently flick the cell strainer with a 1 ml syringe to resuspend the nuclei for passing through the cell strainer.

[0144] Important: The pore size of the cell strainer needs to be adjusted according to the plant species or tissue type.

[0145] 11. After 400 μl of the nuclei solution has been filtered, rinse the 20 μm cell strainer with 100 μl of lx NIB twice, and collect about 500 ~ 600 μl of the nuclei solution.

[0146] 12. Prepare the cell sorting collection tube: pipette 2 ml of cell sorting collection buffer (lx PBS and 1% BSA) into a new 15 ml tube, add 20 μl of RNase inhibitor for collecting the nuclei after flow sorting, and store on ice until use.

[0147] 13. Flow sorting of the nuclei. The flow cytometer used in this protocol is Sony MA900. Adjust the cell sorting parameters: (1) Instrument parameters: turn on 405 and 488 nm lasers, 100 μm sorting chip, FSC channel value = 2.00%, sorting pressure = 2; (2) Sensor gain parameters: FSC value = 7, BSC value = 30.0%. Collect 50,000 ~ 100,000 nuclei events.

[0148] Important: Adjust the parameters for flow sorting of the nuclei according to the plant species and tissue type, including: sensor parameters, sorting pressure, collection area.

[0149] 14. Take 1 μl of the sorted nuclei sample to a fluorescence microscope to observe the sorting results.

[0150] 15. Centrifuge the collected nuclei solution at 1000g for 5 min at 4 °C to collect the nuclei.

[0151] Important: The centrifugation parameters for this step need to be adjusted according to the plant species or tissue type.

[0152] 16. Remove the supernatant, leaving about 50 μl of the volume, resuspend the pellet, and transfer to a 1.5 ml centrifuge tube. Take 1 μl to a new 1.5 ml centrifuge tube, dilute 10-fold, and take 1 μl for observation and counting.

[0153] 17. After counting, take 1-3w of the cell nuclei solution, add 1x PBS with 1% BSA and RNase inhibitor to 43.2 μl, and follow the subsequent single cell nuclei library construction process according to the procedure of 10x Genomics. If more cell nuclei are obtained after sorting, more cell nuclei can be taken for library construction. The maximum is not more than 40,000, and the minimum is not less than 10,000.

[0154] The cell nucleus extraction buffer (1x NIB) used in the above steps is prepared according to Table 1:

[0155] Table 1 Preparation of cell nucleus extraction buffer (1x NIB)

[0156]

[0157] Prepare 10 ml of 1x NIB for each sample.

[0158] The sorting collection buffer used in the above steps is prepared according to the following table

[0159] Table 2 Preparation of sorting collection buffer

[0160]

[0161] Prepare 2 ml of sorting collection buffer for each sample, and add RNase Inhibitor at a ratio of 1:100 before use.

[0162] Example 1 and Comparative Example 1

[0163] Example 1: Using soybean shoot apical meristem, prepare single cell nuclei according to the above steps, and refer to Tables 3 and 4 for specific adjustment parameters.

[0164] Comparative Example 1: Using soybean shoot apical meristem and the single cell nucleus extraction method before optimization. The single cell nucleus extraction method before optimization is different from Example 1 in that the tissue chopping time is more than 5 minutes, the centrifugal parameter is 2000g, and the sorting pressure is 5.

[0165] The comparison of the methods of Example 1 and Comparative Example 1 proves that the integrity of the nuclei after optimization in Example 1 is significantly improved, and the degree of cell adhesion is significantly reduced Figure 1 ).

[0166] Example 2 and Comparative Example 2

[0167] Example 2: Using Leymus mollis seedling tissue, prepare single cell nuclei according to the above steps, and refer to Tables 3 and 4 for specific adjustment parameters.

[0168] Comparative Example 2: Using young seedling tissue of Leymus chinensis and the single cell nucleus extraction method before optimization. The single cell nucleus extraction method before optimization is different from Example 2 in that the tissue chopping time is more than 5 min, the centrifugal parameter is 1000 g, and there is no second nucleus filtering step.

[0169] Example 2 and Comparative Example 2 prove that the integrity of the nucleus after optimization in Example 2 is significantly improved, and the degree of cell adhesion is significantly reduced. Figure 2 ).

[0170] Example 3 and Comparative Example 3

[0171] Example 3: Using root nodule tissue of Sesbania cannabina, single cell nuclei are prepared according to the above steps, and the specific adjusted parameters refer to Table 3 and Table 4.

[0172] Comparative Example 3: Using root nodule tissue of Sesbania cannabina and the single cell nucleus extraction method before optimization. The single cell nucleus extraction method before optimization is different from Example 3 in that the tissue chopping time is more than 5 min, and the centrifugal parameter is 1000 g.

[0173] Example 3 and Comparative Example 3 prove that the integrity of the nucleus after optimization in Example 3 is significantly improved, and the degree of cell adhesion is significantly reduced. Figure 3 ).

[0174] Example 4 and Comparative Example 4

[0175] Example 4: Using root tissue of Sesbania cannabina, single cell nuclei are prepared according to the above steps, and the specific adjusted parameters refer to Table 3 and Table 4.

[0176] Comparative Example 4: Using root tissue of Sesbania cannabina and the single cell nucleus extraction method before optimization. The single cell nucleus extraction method before optimization is different from Example 4 in that the tissue chopping time is more than 5 min, the centrifugal parameter is 500 g, and the sorting pressure is 6.

[0177] Example 4 and Comparative Example 4 prove that the integrity of the nucleus after optimization in Example 4 is significantly improved, and the degree of cell adhesion is significantly reduced. Figure 4 ).

[0178] Example 5 and Comparative Example 5

[0179] Example 5: Using stem tissue of Sesbania cannabina, single cell nuclei are prepared according to the above steps, and the specific adjusted parameters refer to Table 3 and Table 4.

[0180] Comparative Example 5: Using Amaranthus stem tissue and the unoptimized single nucleus extraction method. The unoptimized single nucleus extraction method differs from Example 5 in that the tissue chopping time is more than 5 min, the centrifugation parameter is 500 g, and the sorting pressure is 4.

[0181] Example 5 and Comparative Example 5 demonstrate that the optimized single nucleus extraction method of Example 5 significantly improves the integrity of the nuclei and significantly reduces the degree of cell clumping compared to the method of Comparative Example 5. Figure 5 ).

[0182] Example 6 and Comparative Example 6

[0183] Example 6: Using Amaranthus leaf tissue, single nuclei were prepared according to the above procedure with the specific adjusted parameters referring to Table 3 and Table 4.

[0184] Comparative Example 6: Using Amaranthus leaf tissue and the unoptimized single nucleus extraction method. The unoptimized single nucleus extraction method differs from Example 6 in that the centrifugation parameter is 500 g, there is no second nuclei filtration, and the sorting pressure is 4.

[0185] Example 6 and Comparative Example 6 demonstrate that the optimized single nucleus extraction method of Example 6 significantly improves the integrity of the nuclei and significantly reduces the degree of cell clumping compared to the method of Comparative Example 6. Figure 6 ).

[0186] Example 7 and Comparative Example 7

[0187] Example 7: Using Amaranthus root tip tissue, single nuclei were prepared according to the above procedure with the specific adjusted parameters referring to Table 3 and Table 4.

[0188] Comparative Example 7: Using Amaranthus root tip tissue and the unoptimized single nucleus extraction method. The unoptimized single nucleus extraction method differs from Example 7 in that the tissue chopping time is more than 5 min and the centrifugation parameter is 500 g.

[0189] Example 7 and Comparative Example 7 demonstrate that the optimized single nucleus extraction method of Example 7 significantly improves the integrity of the nuclei and significantly reduces the degree of cell clumping compared to the method of Comparative Example 7. Figure 7 ).

[0190] Table 3. Parameters used in the process of nucleus isolation in different examples

[0191]

[0192] Table 4. Parameters used in the process of nucleus sorting in different examples

[0193]

[0194] The cell nuclei extracted by the method of each example and comparative example were dyed, and the results were as follows Figures 1-7 The quality comparison of the cell nuclei extraction in different plant species and tissues was demonstrated, and the superiority of the method of the present application was verified.

[0195] The technical features of the above-described examples can be combined in any manner. In order to make the description simple, all possible combinations of the technical features in the above-described examples are not described, however, as long as the combinations of the technical features do not contradict, they should be considered as the scope of the present disclosure.

[0196] The above-described examples only express several embodiments of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these are within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for preparing a single nucleus of a plant tissue, characterized by, The preparation method comprises the following steps: (1) adding a nuclear extraction buffer to plant tissue, continuously crushing the plant tissue, and preparing a crushed sample; (2) collecting supernatant from the sample, and collecting nuclei from the supernatant; (3) resuspending the nuclei in the nuclear extraction buffer to prepare a nuclear suspension; and (4) subjecting the nuclear suspension to cell flow sorting to obtain sorted single nuclei; The nuclear extraction buffer comprises the following components at the following working concentrations: NIB 1×, protease inhibitor Cocktail 1×, RNase inhibitor 0.4 U / μL, and DTT 1 mM; The plant tissue is derived from soybean, Leymus chinensis, and Sesbania cannabina.

2. The production method according to claim 1, characterized by, The time for continuously crushing the plant tissue is 2 min ~ 5 min.

3. The production method according to claim 2, characterized by, The plant tissue comprises roots, stems, leaves, and whole plants; Optionally, the plant tissue comprises soybean stem tip meristem, Leymus chinensis seedlings, Sesbania cannabina root nodules, Sesbania cannabina roots, Sesbania cannabina stems, Sesbania cannabina leaves, or Sesbania cannabina root tips.

4. The production method according to any one of claims 1 to 3, characterized by, In step (2), the supernatant is subjected to a first filtration; Optionally, in step (3), after resuspending the nuclei, the nuclei are subjected to a second filtration.

5. The production method according to any one of claims 1 to 3, characterized by, The nuclei are collected by centrifugation at 300×g ~ 1000×g.

6. The production method according to any one of claims 1 to 3, characterized by, The parameters for cell flow sorting comprise one or more of the following conditions: (1) the FSC channel value is 0.5% ~ 2.0%; (2) the sorting pressure is 1 ~ 3; (3) the FSC value is 3 ~ 5; and (4) the BSC value is 27.0% ~ 30.5%.

7. The preparation method according to claim 4, characterized in that, The cell strainer used in the first filtration has a size of 40 μm; and / or the cell strainer used in the second filtration has a size of 20 μm ~ 30 μm.

8. The production method according to claim 7, characterized by, After step (1) is completed, the number and state of the nuclei in the sample are observed; Optionally, when the number of nuclei in the sample is small, the precipitate after centrifugation of the sample is crushed.

9. A single-nucleus transcriptome sequencing method characterized by, The sequencing method comprises the step of preparing single nuclei using the preparation method of any one of claims 1 to 8.

10. The sequencing method of claim 9, wherein, The method further comprises the step of sequencing the single nuclei. The preparation method of any one of claims 1 to 8.

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