A method and application for simultaneously preparing protoplasts and single cell nuclei from Arabidopsis leaf tissue

Abstract

The present invention belongs to the field of biotechnology, and provides a method and application for simultaneously preparing protoplasts and single cell nuclei from Arabidopsis leaf tissue. The method of the present invention can simultaneously obtain protoplasts and cell nuclei of the same plant tissue, and can realize sequencing of protoplasts and cell nuclei of the same plant tissue, thereby obtaining genetic information of plant cells more comprehensively. Currently, no technology for simultaneously preparing protoplasts and extracting single cell nuclei in the same plant tissue has been found. The present invention fills the technical gap of the prior art and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to a method for simultaneously preparing protoplasts and single cell nuclei from Arabidopsis leaf tissue and simultaneously performing single cell sequencing and single cell nucleus sequencing on the same leaf tissue to obtain its genetic information. Background Art

[0002] Single-cell transcriptome analysis is widely used in humans and animal models to reveal the heterogeneity of gene expression between different cell types in tissues or organs. Currently, single-cell sequencing technology has been well applied in tumors, immunity, development and other fields, but plant single-cell research is still in the development stage.

[0003] Single-cell analysis requires the isolation and labeling of messenger RNA (mRNA) from each cell. In the field of plant research, the isolation of model plant protoplasts is now basically stable, but plant single-cell sequencing has certain defects. In the actual application of protoplast isolation, plant tissues have different cell wall thicknesses and vacuolar osmotic pressures with different developmental stages and functions. The enzymatic digestion of plant cell walls, as a stress, easily stimulates plant cells to produce stress responses, induces the expression of some stress-responsive genes, causes partial cell loss, and leads to inequality in cell types in the prepared protoplast population. This limitation is particularly important for cell types located in the inner layer of tissues or the internal tissues of organs, which is not conducive to a comprehensive and in-depth study of the biological processes of plant cell development.

[0004] Plant single-cell nuclear sequencing can make up for some of the shortcomings of plant single-cell sequencing, reduce artificially introduced transcriptional bias, and relatively improve the comprehensiveness of cell types. Since the nuclear membrane is more stable than the cell membrane, the sample types of plant single-cell nuclear sequencing are richer than those of plant single-cell sequencing. Similarly, plant single-cell nuclear sequencing also has certain defects. Plant single-cell nuclear sequencing cannot obtain free mRNA information in the cytoplasm and can only detect mRNA in the nucleus.

[0005] Based on this, plant protoplasts and single cell nuclei are prepared in the same tissue, and single cell sequencing and single cell nuclear sequencing are carried out simultaneously, which is possible to solve the above defects. Taking the model plant Arabidopsis thaliana as an example, the present invention explores an experimental method for obtaining tissue cell types as comprehensively as possible, which plays a key role in comprehensively drawing plant cell maps and in-depth research on plant cell biological functions. Among them, a key technical problem is how to prepare protoplasts and cell nuclei of the same tissue of the plant at the same time, so as to achieve the purpose of carrying out single cell sequencing and single cell nuclear sequencing on the same tissue of the plant at the same time. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and innovatively propose a method for simultaneously preparing protoplasts and single-cell nuclei from Arabidopsis leaf tissue, as well as a method for simultaneously preparing protoplasts and single-cell nuclei and simultaneously performing single-cell sequencing and single-cell nucleus sequencing on the same leaf tissue to obtain its genetic information.

[0007] The present invention provides a method for simultaneously preparing protoplasts and single cell nuclei of the same Arabidopsis tissue and its application. The technical scheme adopted by the present invention is as follows:

[0008] The present invention provides an extraction method for separating protoplasts and single cell nuclei from the same Arabidopsis leaf tissue, comprising the following steps:

[0009] (1) Cut Arabidopsis leaves, rinse them and cut them into thin strips, put them into a culture dish, add WI buffer to grind them, and then transfer them to a culture dish containing enzymatic solution, place them on a constant temperature mixer in the dark for digestion, and obtain digestion solution and digested Arabidopsis leaf tissue.

[0010] (2) Slowly sieving the digestion solution to obtain the sieved supernatant and the digested Arabidopsis leaf tissue, rinsing the remaining tissue with WI buffer, filtering the rinsing solution into the sieved supernatant to obtain a filtrate, which contains protoplasts, and retaining the remaining Arabidopsis leaf tissue.

[0011] (3) The filtrate is centrifuged for the first time, the supernatant is discarded to obtain a precipitate, the precipitate is resuspended in WI buffer and then centrifuged for the second time, the supernatant is discarded to obtain a precipitate, the precipitate is resuspended in WI buffer to obtain a protoplast suspension, and the protoplasts are counted under a microscope.

[0012] (4) The Arabidopsis leaf tissue remaining after digestion in step 2 was transferred to an EP tube, steel balls were added to the tube, and the tube was crushed with liquid nitrogen and then added with lysis solution for lysis.

[0013] (5) Add the washing buffer to the lysate after incubation and sieve it to obtain the sieved supernatant.

[0014] (6) Wash the sieved supernatant by centrifugation twice, discard the supernatant and keep the precipitate, resuspend it in Wash Buffer to obtain a single cell nucleus suspension, and count it under a microscope.

[0015] As a preferred embodiment of the method for extracting Arabidopsis leaf tissue protoplasts and single cell nuclei described in the present invention, in step (1), the enzymatic hydrolyzate contains 2% cellulase RS (Solarbio: C8290-1G), 0.4% (m / v) maceration enzyme R-10 (Solarbio: M8190-1G), 0.5-1% (m / v) xylanase (source leaf: X2753-10G), 8% (m / v) sorbitol (Solarbio: 60319ES76), 20mM MES (PH=5.7) (Sigma: M3671-50G), 20mM KCl (Sigma: P9541-500G), 10mM CaCl2 (Sigma: 93639-100G), 0.1% (v / v) BSA (MACS: 130091376) and sterile water.

[0016] The light-proof digestion conditions are 37° C. and 200 RPM. In addition, the state of the digestion solution and the tissue in the digestion solution should be observed every 20 minutes of the light-proof digestion time. If the tissue sinks to the bottom of the digestion solution and the digestion solution appears turbid, 15 μL of the digestion solution is aspirated, and the number and state of the protoplasts are observed under a microscope, and the total number of protoplasts obtained by enzymatic hydrolysis is calculated.

[0017] The width of the cut tissue slice is 2-4 mm.

[0018] The culture dish is a culture dish with a diameter of 35 mm.

[0019] The WI buffer contains 0.5 M mannitol, 4 mM MES (PH=5.7) and 20 mM KCl.

[0020] As a preferred embodiment of the method for extracting Arabidopsis leaf tissue protoplasts and single cell nuclei described in the present invention, in step (2), a 40 μm cell sieve is used for sieving, and the WI buffer contains 0.5 M mannitol, 4 mM MES (PH=5.7) and 20 mM KCl.

[0021] As a preferred embodiment of the method for extracting protoplasts and single cell nuclei from leaf tissue of Arabidopsis thaliana described in the present invention, in step (3), the centrifugation is carried out at 4°C, 100×g for 7-10 min (preferably 10 min), and the centrifuge lifting rate is 0; the microscopic examination is carried out using 0.4% trypan blue (Thermo Fisher Scientific, T10282) by mass fraction, and the trypan blue solution and the protoplast suspension are mixed at a volume ratio of 1:1. Preferably, 15 μL of trypan blue solution and 15 μL of protoplast suspension are mixed, the number and state of the protoplasts are observed under a microscope, and the total number of protoplasts finally obtained is calculated.

[0022] As a preferred embodiment of the method for extracting protoplasts and single cell nuclei of Arabidopsis leaf tissue described in the present invention, in step (4), the lysis solution contains 45mM MgCl2, 20mM CaCl2, 20mM 3-morpholinepropanesulfonic acid (MOPS), 30mM sodium citrate, 5% (m / v) dextran T-40 (Dextran T40), 1% (w / v) polyvinylpyrrolidone (PVP40), 0.2% (v / v) immunostaining permeabilization solution TritonX-100, 10mM EDTA-Na2, 20μL / mL mercaptoethanol, 0.4U / μL RNase Inhibitor and sterile water; the lysis is carried out on ice for 7 to 10 minutes.

[0023] The number of the steel ball is preferably one.

[0024] The volume of the EP tube is 2 mL.

[0025] As a preferred embodiment of the method for extracting Arabidopsis leaf tissue protoplasts and single cell nuclei described in the present invention, in step (5), the Wash Buffer contains 0.5-1% TritionX-100, 1% BSA, 0.2U / μL RNase inhibitor and PBS; when sieving, first pass through a 40μm pore size cell sieve, and then pass through a 30μm pore size cell sieve.

[0026] As a preferred embodiment of the method for extracting Arabidopsis leaf tissue protoplasts and single cell nuclei described in the present invention, in step (6), the first centrifugation is performed at 300g for 6 to 8 minutes, and the adapted temperature is 4°C; the second centrifugation is performed at 300g for 8 to 10 minutes, and the adapted temperature is 4°C; the microscopic examination is performed using DAPI, and DAPI and the single cell nucleus suspension are mixed at a volume ratio of 1:4. Preferably, 2 μL of DAPI is mixed with 8 μL of the single cell nucleus suspension for microscopic examination.

[0027] The present invention also provides a protoplast suspension and a single cell nucleus suspension prepared by the method. The protoplasts have high purity and low fragmentation rate, and the number of protoplasts reaches 2.8×10 6 ~2.83×10 6 The survival rate of protoplasts was 93% to 94%, the clumping rate was 3%, and the fragmentation rate was 2%. The number of cell nuclei was 1.6×10 6 ~1.65×10 6 The proportion of nuclear agglomerations was 3%, and the proportion of nuclear fragments was 5% to 6%.

[0028] The present invention also proposes the application of the method suitable for simultaneously preparing protoplasts and single cell nuclei from Arabidopsis leaf tissues to simultaneously sequencing the protoplasts and single cell nuclei from Arabidopsis leaf tissues.

[0029] The present invention also provides a kit, which can prepare a protoplast suspension and a single cell nucleus suspension in the same tissue.

[0030] Preferably, the kit can be used for simultaneously sequencing a protoplast suspension and a single cell nucleus suspension prepared from the same Arabidopsis leaf tissue; the kit comprises ① a protoplast hydrolysate, wherein the formula is: 2% RS cellulase, 0.4% (m / v) R-10 macerator, 0.5-1% (m / v) xylanase, 8% (m / v) sorbitol, 20 mM MES (PH=5.7), 20 mM KCl, 1 2. WI buffer, the formula of which is: 0.5M mannitol, 4mM MES (PH = 5.7) and 20mM KCl; 3. Nuclear lysis solution, the formula of which is: 45mM MgCl2, 20mM CaCl2, 20mM 3-morpholinepropanesulfonic acid (MOPS), 30mM sodium citrate, 5% (m / v) dextran T-40 (Dextran T40), 1% (w / v) polyvinyl pyrrolidone (PVP40), 0.2% (v / v) immunostaining permeabilization solution TritonX-100, 10mM EDTA-Na2, 20μL / mL mercaptoethanol and 0.4U / μL RNase Inhibitor; ④ Wash Buffer, whose formula is: 0.5-1% TritionX-100, 1% BSA, 0.2U / μL RNAseinhibitor and PBS.

[0031] Compared with the prior art, the beneficial effects of the present invention include: the present invention uses Arabidopsis leaves as materials, and according to the species characteristics of Arabidopsis and the structural characteristics of the leaves, through appropriate component compounding, the number of protoplasts and cell nuclei prepared is increased, and at the same time, it is beneficial to ensure the integrity and activity of protoplasts and single cell nuclei. The present invention can simultaneously obtain the protoplasts and cell nuclei of Arabidopsis leaves, and then can simultaneously detect the information of single cell transcriptome and mRNA in the nucleus in Arabidopsis leaves. The technical scheme provided by the present invention can obtain more meaningful data in the same leaf tissue and mine more biological information. The method of the present invention can simultaneously obtain the protoplasts and cell nuclei of the same plant tissue, and can realize the sequencing of the protoplasts and cell nuclei of the same plant tissue, and obtain the genetic information of plant cells more comprehensively. At present, no technology for simultaneously preparing protoplasts and extracting single cell nuclei in the same tissue of plants has been found. The present invention fills the technical gap in this aspect of the prior art and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the effect of separating Arabidopsis leaf protoplasts in Example 1 of the present invention.

[0033] Figure 2 This is a diagram showing the effect of separating Arabidopsis leaf nuclei in Example 1 of the present invention.

[0034] Figure 3 This is a diagram showing the effect of Arabidopsis leaf protoplast separation in Example 2 of the present invention.

[0035] Figure 4 This is a diagram showing the effect of separating Arabidopsis leaf nuclei in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further clearly and completely described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. All technical solutions that are identical or similar to the present invention are within the protection scope of the present invention.

[0037] Example 1

[0038] S1. Solution preparation

[0039] The enzymatic solution and WI buffer used for preparing protoplasts and the lysis solution and Wash Buffer used for preparing cell nuclei were prepared. The prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic solution and WI buffer were placed at room temperature for standby use, and the lysis solution and Wash Buffer were placed on ice for standby use. The formulas of the protoplast enzymatic solution and WI buffer are shown in Table A, and the formulas of the cell nucleus lysis solution and Wash Buffer are shown in Table B.

[0040] Table A Protoplast enzymatic solution and WI buffer formula

[0041]

[0042] Table B Cell Nuclear Lysis Solution and Wash Buffer Formula

[0043]

[0044]

[0045] S2. Sample pretreatment

[0046] Take a complete leaf of Arabidopsis thaliana plant, wash the dust on the leaf surface with sterile water, flatten it in a culture dish covered with moist kitchen paper, and use a blade with the help of tweezers to cut the leaf into 2-4 mm filaments. When cutting, be careful to always move the blade from one side to the other, and do not cut back and forth. Prepare tissue slices, then transfer them to a culture dish containing 5 mL of enzymatic solution and seal them with a sealing film.

[0047] Each 5 mL of enzymatic hydrolysate corresponds to 0.5 (±0.01) g of tissue fresh weight.

[0048] S3. Tissue Enzymatic Dissociation

[0049] The culture dish containing the enzymatic solution and tissue slices was placed on a thermomixer and enzymatically digested at 37°C and 200RPM in the dark. When the enzymatic solution treatment time was 1.2h, it was observed that the Arabidopsis leaf tissue was completely dispersed.

[0050] At this time, a portion of the enzymatic hydrolysis solution was aspirated for microscopic examination, the number and state of the protoplasts were observed under a microscope, and the total number of protoplasts obtained by enzymatic hydrolysis was calculated.

[0051] S4. Digestive fluid filtration

[0052] Assemble a 40μm pore cell sieve into a 50mL centrifuge tube, use room temperature WI buffer to fully rinse the cell sieve, draw the digestion solution and add it drop by drop to the sieve, and filter slowly. Add 3mL WI buffer to the remaining tissue in the culture dish, let it stand for 1min, and then filter the rinse solution according to the above method. After filtering, collect and retain the tissue for preparing cell nuclei.

[0053] S5. Centrifugation

[0054] The filtrate was divided into new 15 mL centrifuge tubes, with no more than 5 mL of liquid in each tube. Using a horizontal rotor, centrifuge at 4°C, 100 × g for 10 min with the centrifuge speed set to 0. Carefully aspirate the supernatant and retain the precipitate to enrich the protoplasts.

[0055] S6. Microscopic examination

[0056] Add 1 mL of WI buffer to resuspend the precipitate, aspirate 15 μL of tissue digestion solution, observe the number and state of protoplasts under a microscope, and calculate the total number of enriched protoplasts.

[0057] S7. Protoplast washing

[0058] Add WI buffer to 5 mL, centrifuge in a horizontal rotor, 4°C, 100×g for 7 min, and set the acceleration and deceleration of the centrifuge to 0. Carefully aspirate the supernatant, retain the precipitate, and add 0.5 mL WI buffer to resuspend the precipitate.

[0059] S8. Microscopic examination

[0060] Pipette 15 μL of 0.4% trypan blue solution, mix it with the protoplast suspension at a volume ratio of 1:1, and add it dropwise to the sample well of the hemocytometer. Observe the number and state of the protoplasts under a microscope, count the proportion of surviving protoplasts, the proportion of protoplast agglomerates, the proportion of fragments, and the protoplast concentration, and calculate the total number of protoplasts obtained.

[0061] S9. Lysis of cell nuclei

[0062] Transfer the remaining Arabidopsis leaf tissue after digestion to a 15 mL centrifuge tube, add 2 mL of lysis buffer and mix well, and incubate on ice for 10 min.

[0063] S10.Sieving

[0064] Use Wash Buffer to dilute the above cell nucleus suspension to 8mL system, assemble the 40μm pore size cell sieve into a 50mL centrifuge tube, assemble the 30μm pore size cell sieve into a 15mL centrifuge tube, and use Wash Buffer to fully rinse the cell sieve. The diluted cell nucleus suspension is first passed through a 40μm cell sieve, the sieve is rinsed with Wash Buffer, and then passed through a 30μm cell sieve, the sieve is rinsed with Wash Buffer, and the cell nucleus suspension system reaches 10mL after sieving.

[0065] S11. Washing

[0066] Place the cell nucleus suspension in a 4°C low-temperature centrifuge, centrifuge at 300g for 6 minutes, and discard the supernatant. Resuspend the pellet with 3mL of Wash Buffer, centrifuge at 4°C and 300g for 10 minutes, discard the supernatant, and resuspend the pellet with 500μL Wash Buffer.

[0067] S12. Secondary filtration and washing

[0068] Add PBS buffer to make up the cell nucleus suspension to 3 mL, pass through the MACS MS separation column, and filter out impurities. Centrifuge the filtrate again at 4°C, 300g for 10 min, discard the supernatant, and resuspend the pellet in 50 μL Wash Buffer.

[0069] S13. Microscopic examination

[0070] Pipette 2 μL of DAPI staining solution and 8 μL of cell nucleus suspension, mix well and add drops into the sample well of the hemocytometer. Observe the number and state of cell nuclei under a microscope, count the agglomeration ratio, fragment ratio and cell nucleus concentration of the cell nuclei, and calculate the final total number of cell nuclei.

[0071] S14. Results and Analysis

[0072] The experimental results showed that Arabidopsis leaves were hydrolyzed with enzymatic solution, protoplasts were stained with trypan blue and counted with a hemocytometer. The number of protoplasts was 2.8×10 6 The proportion of surviving protoplasts was 93%, the proportion of protoplast agglomeration was 3%, and the proportion of fragments was 2%. The number of cell nuclei was 1.6×10 by DAPI staining and hemocytometer counting. 6 The percentage of nuclear agglomeration was 3%, and the percentage of nuclear fragments was 6%; both met the sample quality requirements for single-cell and single-nucleus sequencing on the 10x Genomics platform.

[0073] Example 2

[0074] S1. Solution preparation

[0075] The enzymatic solution and WI buffer used for preparing protoplasts and the lysate and Wash Buffer used for preparing cell nuclei were prepared. The prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic solution and WI buffer were placed at room temperature for standby use, and the lysate and Wash Buffer were placed on ice for standby use. The formulas of the protoplast enzymatic solution and WI buffer are shown in Table C, and the formulas of the cell nucleus lysate and Wash Buffer are shown in Table D.

[0076] Table C Protoplast enzymatic solution and WI buffer formula

[0077]

[0078]

[0079] Table D: Cell Nuclear Lysis Solution and Wash Buffer Formula

[0080]

[0081] S2. Sample pretreatment

[0082] Take a complete leaf of Arabidopsis thaliana plant, wash the dust on the leaf surface with sterile water, flatten it in a culture dish covered with moist kitchen paper, and use a blade with the help of tweezers to cut the leaf into 2-4 mm filaments. When cutting, be careful to always move the blade from one side to the other, and do not cut back and forth. Prepare tissue slices, then transfer them to a culture dish containing 5 mL of enzymatic solution and seal them with a sealing film.

[0083] Each 5 mL of enzymatic hydrolysate corresponds to 0.5 (±0.01) g of tissue fresh weight.

[0084] S3. Tissue Enzymatic Dissociation

[0085] The culture dish containing the enzymatic solution and tissue slices was placed on a thermomixer and enzymatically digested at 37°C and 200RPM in the dark. When the enzymatic solution treatment time was 1.2h, it was observed that the Arabidopsis leaf tissue was completely dispersed.

[0086] At this time, a portion of the enzymatic hydrolysis solution was aspirated for microscopic examination, the number and state of the protoplasts were observed under a microscope, and the total number of protoplasts obtained by enzymatic hydrolysis was calculated.

[0087] S4. Digestive fluid filtration

[0088] Assemble a 40μm pore cell sieve into a 50mL centrifuge tube, use room temperature WI buffer to fully rinse the cell sieve, draw the digestion solution and add it drop by drop to the sieve, and filter slowly. Add 3mL WI buffer to the remaining tissue in the culture dish, let it stand for 1min, and then filter the rinse solution according to the above method. After filtering, collect and retain the tissue for preparing cell nuclei.

[0089] S5. Centrifugation

[0090] The filtrate was divided into new 15 mL centrifuge tubes, with no more than 5 mL of liquid in each tube. Using a horizontal rotor, centrifuge at 4°C, 100 × g for 10 min with the centrifuge speed set to 0. Carefully aspirate the supernatant and retain the precipitate to enrich the protoplasts.

[0091] S6. Microscopic examination

[0092] Add 1 mL of WI buffer to resuspend the precipitate, aspirate 15 μL of tissue digestion solution, observe the number and state of protoplasts under a microscope, and calculate the total number of enriched protoplasts.

[0093] S7. Protoplast washing

[0094] Add WI buffer to 5 mL, centrifuge in a horizontal rotor, 4°C, 100×g for 10 min, and set the acceleration and deceleration of the centrifuge to 0. Carefully aspirate the supernatant, retain the precipitate, and add 0.5 mL WI buffer to resuspend the precipitate.

[0095] S8. Microscopic examination

[0096] Pipette 15 μL of 0.4% trypan blue solution, mix it with the protoplast suspension at a volume ratio of 1:1, and add it dropwise to the sample well of the hemocytometer. Observe the number and state of the protoplasts under a microscope, count the proportion of surviving protoplasts, the proportion of protoplast agglomerates, the proportion of fragments, and the protoplast concentration, and calculate the total number of protoplasts obtained.

[0097] S9. Lysis of cell nuclei

[0098] Transfer the remaining Arabidopsis leaf tissue after digestion to a 15 mL centrifuge tube, add 2 mL of lysis buffer and mix well, and incubate on ice for 10 min.

[0099] S10.Sieving

[0100] Use Wash Buffer to dilute the above cell nucleus suspension to 8mL system, assemble the 40μm pore size cell sieve into a 50mL centrifuge tube, assemble the 30μm pore size cell sieve into a 15mL centrifuge tube, and use Wash Buffer to fully rinse the cell sieve. The diluted cell nucleus suspension is first passed through a 40μm cell sieve, the sieve is rinsed with Wash Buffer, and then passed through a 30μm cell sieve, the sieve is rinsed with Wash Buffer, and the cell nucleus suspension system reaches 10mL after sieving.

[0101] S11. Washing

[0102] Place the cell nucleus suspension in a 4℃ low-temperature centrifuge, centrifuge at 300g for 8min, and discard the supernatant. Resuspend the pellet with 3mL of Wash Buffer, centrifuge at 4℃ and 300g for 10min, discard the supernatant, and resuspend the pellet with 500μL Wash Buffer.

[0103] S12. Secondary filtration and washing

[0104] Add PBS buffer to make up the cell nucleus suspension to 3 mL, pass through the MACS MS separation column, and filter out impurities. Centrifuge the filtrate again at 4°C, 300g for 10 min, discard the supernatant, and resuspend the pellet in 50 μL Wash Buffer.

[0105] S13. Microscopic examination

[0106] Pipette 2 μL of DAPI staining solution and 8 μL of cell nucleus suspension, mix well and add drops into the sample well of the hemocytometer. Observe the number and state of cell nuclei under a microscope, count the agglomeration ratio, fragment ratio and cell nucleus concentration of the cell nuclei, and calculate the final total number of cell nuclei.

[0107] S14. Results and Analysis

[0108] The experimental results showed that Arabidopsis leaves were hydrolyzed with enzymatic solution, protoplasts were stained with trypan blue and counted with a hemocytometer. The number of protoplasts was 2.83×10 6 The proportion of surviving protoplasts was 94%, the proportion of protoplast agglomeration was 3%, and the proportion of fragments was 2%. The number of cell nuclei was 1.65×10 by DAPI staining and hemocytometer counting. 6 The percentage of nuclear agglomeration was 3%, and the percentage of nuclear fragments was 5%; both met the sample quality requirements for single-cell and single-nucleus sequencing on the 10×Genomics platform.

[0109] Comparative Example 1

[0110] In this example, the cell wall was removed by grinding to explore the simultaneous preparation of protoplasts and single-cell nuclear suspensions from the same Arabidopsis leaf tissue. Protoplast enrichment was performed with reference to "An efficient and universal protoplast isolation protocol suitable for transient gene expression analysis and single-cell RNA sequencing"; single-cell nuclear suspension preparation was performed with reference to "Single-nucleus RNA sequencing of plant tissues using a nanowell-based system".

[0111] S1. Solution preparation

[0112] PB buffer and W5 buffer for preparing protoplasts and Wash Buffer for preparing cell nuclei were prepared. All prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic solution and WI buffer were kept at room temperature for later use. The Wash Buffer was kept on ice for later use. Its formula is shown in Table 1.

[0113] Table 1 Protoplast PB buffer, W5 buffer and nucleus Wash Buffer formula

[0114]

[0115]

[0116] S2. Sample pretreatment

[0117] Take a whole leaf of Arabidopsis thaliana plant, wash the dust on the surface of the leaf with sterile water, and cut it into pieces with scissors.

[0118] S3. Blade grinding

[0119] Place the cut Arabidopsis leaves in a petri dish, grind manually for 1 min, add 5 mL of PB buffer, and let stand for 10 min. Take part of the buffer for microscopic examination, observe the number and state of protoplasts under a microscope, and calculate the total number of protoplasts obtained by enzymatic hydrolysis.

[0120] S4. Filtering

[0121] Assemble a cell sieve with a pore size of 40 μm into a 50 mL centrifuge tube, use room temperature W5 buffer to fully rinse the cell sieve, use a wide-mouth disposable dropper to absorb the solution in step S3, let it stand, add it drop by drop to the sieve, and filter slowly. Add 3 mL of W5 buffer to the remaining tissue in the culture dish, let it stand for 1 minute, filter the rinse solution according to the above method, collect the tissue after filtration, and store it at 4°C for preparing cell nuclei.

[0122] S5. Centrifugation

[0123] The filtrate was divided into new 15 mL centrifuge tubes, with no more than 5 mL of liquid in each tube. Using a horizontal rotor, centrifuge at 200 × g for 10 min at room temperature (25°C). The supernatant was carefully aspirated and the precipitate was retained to enrich the protoplasts.

[0124] S6. Microscopic examination

[0125] Add 0.1mL W5 buffer to resuspend the precipitate, draw 15μL of 0.4% trypan blue solution, mix it with the protoplast suspension at a volume ratio of 1:1, and drop it into the sample well of the blood cell counting plate. Observe the number and state of protoplasts under a microscope, count the proportion of surviving protoplasts, the proportion of protoplast agglomeration, the proportion of fragments and the concentration of protoplasts, and calculate the total number of protoplasts obtained. Dead protoplasts will be stained blue under an optical microscope, while surviving protoplasts will not be marked by trypan blue.

[0126] S7. Grinding of retained tissue

[0127] The retained tissue in step S4 was taken and ground for another 3 min, 5 mL of Wash Buffer was added, and the mixture was incubated on ice for 10 min.

[0128] S8.Sieving

[0129] Use Wash Buffer to dilute the above cell nucleus suspension to 8mL system, assemble the 70μm pore size cell sieve into a 50mL centrifuge tube, and use Wash Buffer to fully rinse the cell sieve. The diluted cell nucleus suspension is passed through a 70μm cell sieve, and the sieve is rinsed with Wash Buffer to make the cell nucleus suspension system reach 10mL after sieving.

[0130] S9. Washing

[0131] Place the cell nucleus suspension in a 4℃ low-temperature centrifuge, centrifuge at 1000g for 6 minutes, and discard the supernatant. Resuspend the pellet with 3mL of Wash Buffer, centrifuge at 4℃ and 300g for 10 minutes, discard the supernatant, and resuspend the pellet with 500μL Wash Buffer.

[0132] S10. Secondary filtration and washing

[0133] Add PBS buffer to make up the cell nucleus suspension to 3 mL, pass through the MACS MS separation column, and filter out impurities. Centrifuge the filtrate again at 4°C, 1000g for 10 min, discard the supernatant, and resuspend the pellet in 50 μL Wash Buffer.

[0134] S11. Microscopic examination

[0135] Pipette 2 μL of DAPI staining solution and 8 μL of cell nucleus suspension, mix well and add drops into the sample well of the hemocytometer. Observe the number and state of cell nuclei under a microscope, count the agglomeration ratio, fragment ratio and cell nucleus concentration of the cell nuclei, and calculate the final total number of cell nuclei.

[0136] S12. Results and Analysis

[0137] The experimental results showed that the Arabidopsis leaves were ground, the protoplasts were stained with trypan blue and counted with a hemocytometer, the number of protoplasts was 2000, the proportion of surviving protoplasts was 25%, the proportion of protoplast clumping was 15%, and the proportion of fragments was 35%; the nuclei were stained with DAPI and counted with a hemocytometer, the number of nuclei was 5000, the proportion of nuclei clumping was 46%, and the proportion of nuclei fragments was 58%; both failed to meet the sample quality requirements for single-cell and single-cell nucleus sequencing on the 10x Genomics platform.

[0138] Comparative Example 2

[0139] In this example, the cell wall was first removed by enzymatic hydrolysis and then the cell membrane was broken to explore the simultaneous preparation of protoplasts and single nucleus suspensions from the same Arabidopsis leaf tissue. The protoplast preparation method was from "Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis", and the nucleus preparation was carried out with reference to "Single-nucleus RNA sequencing of plant tissues using a nanowell-based system".

[0140] S1. Solution preparation

[0141] The enzymatic solution, WI buffer and Wash Buffer used for preparing protoplasts and cell nuclei were prepared. The prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic solution and WI buffer were placed at room temperature for later use, and the Wash Buffer was placed on ice for later use. The formula is shown in Table 2.

[0142] Table 2 Protoplast enzymatic solution, WI buffer and cell nucleus Wash Buffer formula

[0143]

[0144] S2. Sample pretreatment

[0145] Take a complete leaf of Arabidopsis thaliana plant, wash the dust on the leaf surface with sterile water, flatten it in a culture dish covered with moist kitchen paper, and use a blade to cut the leaf into 0.5 mm filaments with the help of tweezers to prepare tissue slices, which are then transferred to a culture dish containing 5 mL of enzymatic solution and sealed with a sealing film.

[0146] Each 5 mL of enzymatic hydrolysate corresponds to 0.5 (±0.01) g of tissue fresh weight.

[0147] S3. Tissue Enzymatic Dissociation

[0148] The culture dish containing the enzymatic solution and the tissue slices was placed on a thermomixer and enzymatically digested at 37°C and 200RPM in the dark. When the enzymatic solution treatment time was 1 hour, it was observed that the Arabidopsis leaf tissue was completely dispersed.

[0149] At this time, a portion of the enzymatic hydrolysis solution was aspirated for microscopic examination, the number and state of the protoplasts were observed under a microscope, and the total number of protoplasts obtained by enzymatic hydrolysis was calculated.

[0150] S4. Digestive fluid filtration

[0151] Assemble a cell sieve with a pore size of 40 μm into a 50 mL centrifuge tube, use room temperature WI buffer to fully rinse the cell sieve, draw the digestion solution and add it drop by drop to the sieve, and filter slowly. Add 3 mL of WI buffer to the remaining tissue in the culture dish, let it stand for 1 min, and then filter the rinse solution according to the above method. After filtration, collect the tissue and store it at 4°C for preparation of cell nuclei.

[0152] S5. Centrifugation

[0153] The filtrate was divided into new 15 mL centrifuge tubes, with no more than 5 mL of liquid in each tube. Using a horizontal rotor, centrifuge at 100 x g for 10 min at room temperature (25°C). The supernatant was carefully aspirated and the precipitate was retained to enrich the protoplasts.

[0154] S6. Microscopic examination

[0155] Add 1 mL of WI buffer to resuspend the precipitate, aspirate 15 μL of tissue digestion solution, observe the number and state of protoplasts under a microscope, and calculate the total number of enriched protoplasts.

[0156] S7. Centrifugal washing

[0157] Add WI buffer to 5 mL, centrifuge and wash in a horizontal rotor at 25°C, 100×g for 7 min, carefully aspirate the supernatant, retain the precipitate, and add 0.5 mL WI buffer to resuspend the precipitate.

[0158] S8. Microscopic examination

[0159] Pipette 15 μL of 0.4% trypan blue solution, mix it with the protoplast suspension at a volume ratio of 1:1, and add it dropwise to the sample well of the hemocytometer. Observe the number and state of the protoplasts under a microscope, count the proportion of surviving protoplasts, the proportion of protoplast agglomerates, the proportion of fragments, and the protoplast concentration, and calculate the total number of protoplasts obtained.

[0160] S9. Grinding of retained tissue

[0161] The retained tissue in step S4 was taken and ground for another 3 min, 5 mL of Wash Buffer was added, and the mixture was incubated on ice for 10 min.

[0162] S10.Sieving

[0163] Use Wash Buffer to dilute the above cell nucleus suspension to 8mL system, assemble the 70μm pore size cell sieve into a 50mL centrifuge tube, and use Wash Buffer to fully rinse the cell sieve. The diluted cell nucleus suspension is passed through a 70μm cell sieve, and the sieve is rinsed with Wash Buffer to make the cell nucleus suspension system reach 10mL after sieving.

[0164] S11. Centrifugation

[0165] Place the cell nucleus suspension in a 4℃ low-temperature centrifuge, centrifuge at 1000g for 6 minutes, and discard the supernatant. Resuspend the pellet with 3mL of Wash Buffer, centrifuge at 4℃ and 300g for 10 minutes, discard the supernatant, and resuspend the pellet with 500μL Wash Buffer.

[0166] S12. Filtration

[0167] Add Wash Buffer to make up the cell nucleus suspension to 3 mL, pass through the MACS MS column, and filter out impurities. Centrifuge the filtrate again at 4°C, 1000g for 10 min, discard the supernatant, and resuspend the pellet in 50 μL Wash Buffer.

[0168] S13. Microscopic examination

[0169] Pipette 2 μL of DAPI staining solution and 8 μL of cell nucleus suspension, mix well and add drops into the sample well of the hemocytometer. Observe the number and state of cell nuclei under a microscope, count the agglomeration ratio, fragment ratio and cell nucleus concentration of the cell nuclei, and calculate the final total number of cell nuclei.

[0170] S14. Results and Analysis

[0171] The experimental results showed that Arabidopsis leaves were enzymatically hydrolyzed, protoplasts were stained with trypan blue, and counted with a hemocytometer. The number of protoplasts was 1.45×10 6 The remaining tissue after digestion was ground, the nuclei were stained with DAPI and counted on a hemocytometer. The number of nuclei was 4,000, the nuclei clustering ratio was 48%, and the nuclei fragment ratio was 50%. Protoplasts basically meet the sample quality requirements for single-cell sequencing on the 10xGenomics platform. However, single-cell nuclear suspension cannot meet the sample quality requirements for single-cell nuclear sequencing on the 10x Genomics platform.

[0172] Comparative Example 3

[0173] In this example, the method of first enzymatically removing the cell wall and then lysing the cell membrane with chemical reagents was used to explore the simultaneous preparation of protoplasts and single-cell nucleus suspensions from the same Arabidopsis leaf tissue. The method for preparing protoplasts is from "Arabidopsis thaliana protoplasts: a versatile cell system for transient gene expression analysis", and the method for preparing single-cell nucleus suspensions is based on "Single-nucleus RNA sequencing of plant tissues using a nanowell-based system".

[0174] S1. Solution preparation

[0175] The enzymatic hydrolysate and WI buffer for preparing protoplasts and the lysis solution and Wash Buffer for preparing cell nuclei were prepared. The prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic hydrolysate and WI buffer were placed at room temperature for use, and the lysis solution and Wash Buffer were placed on ice for use. The formulas are shown in Table 3.

[0176] Table 3 Protoplast enzymatic solution, WI buffer, nuclear lysis solution, Wash Buffer formula

[0177]

[0178]

[0179] S2. Sample pretreatment

[0180] Take a complete leaf of Arabidopsis thaliana plant, wash the dust on the leaf surface with sterile water, flatten it in a culture dish covered with moist kitchen paper, and use a blade with the help of tweezers to cut the leaf into 0.5 mm filaments. When cutting, be careful to always move the blade from one side to the other, and do not cut back and forth. Prepare tissue slices, then transfer them to a culture dish containing 5 mL of enzymatic solution and seal them with a sealing film.

[0181] Each 5 mL of enzymatic hydrolysate corresponds to 0.5 (±0.01) g of tissue fresh weight.

[0182] S3. Tissue Enzymatic Dissociation

[0183] The culture dish containing the enzymatic solution and the tissue slices was placed on a thermomixer and enzymatically digested at 37°C and 200RPM in the dark. When the enzymatic solution treatment time was 1 hour, it was observed that the Arabidopsis leaf tissue was completely dispersed.

[0184] At this time, a portion of the enzymatic hydrolysis solution was aspirated for microscopic examination, the number and state of the protoplasts were observed under a microscope, and the total number of protoplasts obtained by enzymatic hydrolysis was calculated.

[0185] S4. Digestive fluid filtration

[0186] Assemble a 40μm pore cell sieve into a 50mL centrifuge tube, use room temperature WI buffer to fully rinse the cell sieve, draw the digestion solution and add it drop by drop to the sieve, and filter slowly. Add 3mL WI buffer to the remaining tissue in the culture dish, let it stand for 1min, and then filter the rinse solution according to the above method. After filtering, collect and retain the tissue for preparing cell nuclei.

[0187] S5. Centrifugation

[0188] The filtrate was divided into new 15 mL centrifuge tubes, with no more than 5 mL of liquid in each tube. Using a horizontal rotor, centrifuge at 100 x g for 10 min at room temperature (25°C) with an acceleration of 9. Carefully aspirate the supernatant and retain the precipitate to enrich the protoplasts.

[0189] S6. Microscopic examination

[0190] Add 1 mL of WI buffer to resuspend the precipitate, aspirate 15 μL of tissue digestion solution, observe the number and state of protoplasts under a microscope, and calculate the total number of enriched protoplasts.

[0191] S7. Protoplast washing

[0192] Add WI buffer to 5 mL, centrifuge and wash in a horizontal rotor at 25°C, 100xg for 7 min, carefully aspirate the supernatant, retain the precipitate, and add 0.5 mL WI buffer to resuspend the precipitate.

[0193] S8. Microscopic examination

[0194] Pipette 15 μL of 0.4% trypan blue solution, mix it with the protoplast suspension at a volume ratio of 1:1, and add it dropwise to the sample well of the hemocytometer. Observe the number and state of the protoplasts under a microscope, count the proportion of surviving protoplasts, the proportion of protoplast agglomerates, the proportion of fragments, and the protoplast concentration, and calculate the total number of protoplasts obtained.

[0195] S9. Lysis of cell nuclei

[0196] Transfer the remaining Arabidopsis leaf tissue after digestion to a 15 mL centrifuge tube, add 2 mL of lysis buffer and mix well, and incubate on ice for 10 min.

[0197] S10.Sieving

[0198] Use Wash Buffer to dilute the above cell nucleus suspension to 8mL system, assemble the 70μm pore size cell sieve into a 50mL centrifuge tube, and use Wash Buffer to fully rinse the cell sieve. The diluted cell nucleus suspension is passed through a 70μm cell sieve, and the sieve is rinsed with Wash Buffer to make the cell nucleus suspension system reach 10mL after sieving.

[0199] S11. Washing

[0200] Place the cell nucleus suspension in a 4℃ low-temperature centrifuge, centrifuge at 1000g for 6 minutes, and discard the supernatant. Resuspend the pellet with 3mL of Wash Buffer, centrifuge at 4℃ and 300g for 10 minutes, discard the supernatant, and resuspend the pellet with 500μL Wash Buffer.

[0201] S12. Secondary filtration and washing

[0202] Add PBS buffer to make up the cell nucleus suspension to 3 mL, pass through the MACS MS separation column, and filter out impurities. Centrifuge the filtrate again at 4°C, 1000g for 10 min, discard the supernatant, and resuspend the pellet in 50 μL Wash Buffer.

[0203] S13. Microscopic examination

[0204] Pipette 2 μL of DAPI staining solution and 8 μL of cell nucleus suspension, mix well and add drops into the sample well of the hemocytometer. Observe the number and state of cell nuclei under a microscope, count the agglomeration ratio, fragment ratio and cell nucleus concentration of the cell nuclei, and calculate the final total number of cell nuclei.

[0205] S14. Results and Analysis

[0206] The experimental results showed that Arabidopsis leaves were hydrolyzed with enzymatic solution, protoplasts were stained with trypan blue and counted with a hemocytometer. The number of protoplasts was 1.5×10 6 The proportion of surviving protoplasts was 80%, the proportion of protoplast agglomerates was 6%, and the proportion of fragments was 8%. The remaining tissue after digestion was ground, and the cell nuclei were stained with DAPI and counted with a hemocytometer. The number of cell nuclei was 3×10 4 The protoplasts basically meet the quality requirements of the single-cell sequencing of the 10xGenomics platform for samples, and the number, fragment ratio and agglomeration ratio of the single-cell nuclear suspension are qualitatively improved compared with those of Comparative Examples 1 to 2.

[0207] Comparative Example 4

[0208] In this example, the cutting width of Arabidopsis leaf tissue is adjusted to 0.5-1 mm, and the remaining operations are consistent with those in Comparative Example 3.

[0209] The experimental results are shown in Table 7.

[0210] Comparative Example 5

[0211] In this example, the cutting width of Arabidopsis leaf tissue is adjusted to 2-4 mm, and the remaining operations are consistent with those in Comparative Example 3.

[0212] The experimental results are shown in Table 7.

[0213] Comparative Example 6

[0214] In this example, the cutting width of Arabidopsis leaf tissue is adjusted to 4-6 mm, and the remaining operations are consistent with those in Comparative Example 3.

[0215] The experimental results are shown in Table 7.

[0216] Comparative Example 7

[0217] In this example, on the basis of the cutting width of Arabidopsis leaf tissue being 2-4 mm, the enzymatic hydrolysis time was extended to 120 min, and the remaining operations were consistent with those in Comparative Example 3.

[0218] The experimental results are shown in Table 7.

[0219] Comparative Example 8

[0220] In this example, based on the cutting width of Arabidopsis leaf tissue being 2-4 mm, the concentration of cellulase R-10 in the enzymatic hydrolysate was adjusted to 4%, and the concentration of cleavage enzyme was adjusted to 0.8%. The remaining operations were consistent with those in Comparative Example 3.

[0221] The experiment is shown in Table 7.

[0222] Comparative Example 9

[0223] In this example, the components in the protoplast hydrolysate were based on those shown in Table 3, with the addition of 1% (m / v) pectinase, and the remaining operations were consistent with those in Comparative Example 5.

[0224] The experiment is shown in Table 7.

[0225] Comparative Example 10

[0226] In this example, the components in the protoplast hydrolysate were based on those shown in Table 3, and 2% (m / v) of hemicellulase was additionally added. The remaining operations were consistent with those in Comparative Example 5.

[0227] The experiment is shown in Table 7.

[0228] Comparative Example 11

[0229] In this example, the components in the protoplast hydrolysate were based on those shown in Table 3, with the addition of 0.4% (m / v) of lysase, and the remaining operations were consistent with those in Comparative Example 5.

[0230] The experimental results are shown in Table 7.

[0231] Comparative Example 12

[0232] In this example, the components in the protoplast hydrolysate were based on those shown in Table 3, and 1% (m / v) xylanase was additionally added. The remaining operations were consistent with those in Comparative Example 5.

[0233] The experimental results are shown in Table 7.

[0234] Comparative Example 13

[0235] In this example, the components in the protoplast hydrolysate were based on those shown in Table 3, with the addition of 2% (m / v) snail enzyme, and the remaining operations were consistent with those in Comparative Example 5.

[0236] The experimental results are shown in Table 7.

[0237] Comparative Example 14

[0238] In this example, 2% (m / v) of cellulase R-10 in the protoplast hydrolysate was replaced with 2% (m / v) of cellulase RS, and the remaining operations were consistent with those in Comparative Example 5.

[0239] The experimental results are shown in Table 7.

[0240] Comparative Example 15

[0241] In this example, the protoplast hydrolysate group was additionally added with 1% (m / v) xylanase on the basis of Example 14, and the rest of the operations were consistent with those in Comparative Example 3.

[0242] The experimental results are shown in Table 7.

[0243] Comparative Example 16

[0244] In this example, the concentration of xylanase in the protoplast hydrolysate of Comparative Example 15 was adjusted to 0.2% (m / v), and the remaining operations were consistent with those of Comparative Example 3.

[0245] The experimental results are shown in Table 7.

[0246] Comparative Example 17

[0247] In this example, the concentration of xylanase in the protoplast hydrolysate of Comparative Example 15 was adjusted to 0.5% (m / v), and the remaining operations were consistent with those of Comparative Example 3.

[0248] The experimental results are shown in Table 7.

[0249] Comparative Example 18

[0250] In this example, the concentration of xylanase in the protoplast hydrolysate of Comparative Example 15 was adjusted to 2% (m / v), and the remaining operations were consistent with those of Comparative Example 3.

[0251] The experimental results are shown in Table 7.

[0252] Comparative Example 19

[0253] In this example, the mannitol in the protoplast hydrolysate was replaced with 8% sorbitol, and the remaining operations were consistent with those in Comparative Example 15.

[0254] The experimental results are shown in Table 7.

[0255] Comparative Example 20

[0256] In this example, the sorbitol concentration in Comparative Example 19 was adjusted to 4%, and the remaining operations were consistent with those in Comparative Example 19.

[0257] The experimental results are shown in Table 7.

[0258] Comparative Example 21

[0259] In this example, the sorbitol concentration in Comparative Example 19 was adjusted to 12%, and the remaining operations were consistent with those in Comparative Example 19.

[0260] The experimental results are shown in Table 7.

[0261] Comparative Example 22

[0262] This example explores the effect of temperature on the preparation of Arabidopsis leaf tissue protoplasts.

[0263] S1. Solution preparation

[0264] The enzymatic solution and WI buffer for preparing protoplasts and the lysis solution and Wash Buffer for preparing cell nuclei were prepared. The prepared solutions were filtered through a 0.22 μm filter membrane and placed in a 50 mL centrifuge tube. The enzymatic solution and WI buffer were placed at room temperature for use, and the lysis solution and Wash Buffer were placed on ice for use. The formulas are shown in Table 4.

[0265] Table 4 Protoplast enzymatic solution, WI buffer and nuclear lysis solution, Wash Buffer formula

[0266]

[0267]

[0268] Except that the filtration of the digestion solution was carried out on ice and the temperature during protoplast enrichment and washing was 4°C, the remaining operations were consistent with Comparative Example 21.

[0269] The experimental results are shown in Table 7.

[0270] Comparative Example 23

[0271] This example investigates the effect of the centrifuge lifting speed during centrifugation in the protoplast enrichment and washing steps on the preparation of Arabidopsis leaf tissue protoplasts. The centrifuge lifting speed is set to 5, and the remaining operations are consistent with Comparative Example 22.

[0272] The experimental results are shown in Table 7.

[0273] Comparative Example 24

[0274] This example investigates the effect of the centrifuge lifting speed during centrifugation in the protoplast enrichment and washing steps on the preparation of Arabidopsis leaf tissue protoplasts. The lifting speed during centrifugation is set to 0, and the remaining operations are consistent with Comparative Example 22.

[0275] The experimental results are shown in Table 7.

[0276] Comparative Example 25

[0277] In this example, the centrifugation time in the protoplast washing step was adjusted to 10 min, and the remaining operations were consistent with Comparative Example 24.

[0278] The experimental results are shown in Table 7.

[0279] Comparative Example 26

[0280] In this example, the centrifugation time in the protoplast washing step was adjusted to 15 min, and the remaining operations were consistent with Comparative Example 24.

[0281] The experimental results are shown in Table 7.

[0282] Comparative Example 27

[0283] This example explores the effect of the treatment of the remaining tissue after enzymatic hydrolysis on the preparation of single nuclei of Arabidopsis leaf tissue. Except for the adjustment of the digestion and lysis of the nuclei, the remaining Arabidopsis leaf tissue after digestion was transferred to a 2mL Ep tube, 0.2mL of nucleus lysis solution was added, and the mixture was crushed at 1200rpm for 30s in a normal temperature crusher, and the lysis solution was added to 2mL and mixed, and the mixture was placed on ice for incubation and lysis for 10min. All other operations were consistent with Comparative Example 24.

[0284] The experimental results are shown in Table 7.

[0285] Comparative Example 28

[0286] This example explores the effect of the treatment of the remaining tissue after enzymatic hydrolysis on the preparation of single nuclei of Arabidopsis leaf tissue. Except for the adjustment of the digestion and lysis of the nuclei, the remaining Arabidopsis leaf tissue after digestion was transferred to a 2mL Ep tube, a steel ball was added to the tube, and after liquid nitrogen crushing, 2mL of lysis solution was added to mix, and placed on ice for incubation and lysis for 10 minutes. All other operations were consistent with Comparative Example 24.

[0287] The experimental results are shown in Table 7.

[0288] Comparative Example 29

[0289] In this example, 2.5% (w / v) Ficoll 400 in the cell nuclear lysate was replaced with 1% (w / v) PVP40, and all other operations were consistent with those in Comparative Example 28.

[0290] The experimental results are shown in Table 7.

[0291] Comparative Example 30

[0292] In this example, the concentration of PVP40 in the cell nuclear lysate was adjusted to 0.2% (w / v), and all other operations were consistent with those in Comparative Example 29.

[0293] The experimental results are shown in Table 7.

[0294] Comparative Example 31

[0295] In this example, the concentration of PVP40 in the cell nuclear lysate was adjusted to 2% (w / v), and all other operations were consistent with Comparative Example 29.

[0296] The experimental results are shown in Table 7.

[0297] Comparative Example 32

[0298] In this example, 10 mM Tris-HCl (pH=7.4) in the cell nuclear lysate was replaced with 10 mM MOPS and 20 mM sodium citrate, and all other operations were consistent with Comparative Example 29.

[0299] The experimental results are shown in Table 7.

[0300] Comparative Example 33

[0301] In this example, MOPS and sodium citrate are adjusted to 20 mM and 30 mM respectively, and all other operations are consistent with Comparative Example 29.

[0302] The experimental results are shown in Table 7.

[0303] Comparative Example 34

[0304] In this example, MOPS and sodium citrate are adjusted to 30 mM and 40 mM respectively, and all other operations are consistent with Comparative Example 29.

[0305] The experimental results are shown in Table 7.

[0306] Comparative Example 35

[0307] In this example, DTT in the cell nuclear lysate was replaced with ascorbic acid, and all other operations were consistent with those in Comparative Example 33.

[0308] The experimental results are shown in Table 7.

[0309] Comparative Example 36

[0310] In this example, the DTT in the cell nuclear lysate was replaced with 20 μL / mL of mercaptoethanol, and all other operations were consistent with those in Comparative Example 33.

[0311] The experimental results are shown in Table 7.

[0312] Comparative Example 37

[0313] In this example, the amount of mercaptoethanol is adjusted to 10 μL / mL, and all other operations are consistent with those in Comparative Example 36.

[0314] The experimental results are shown in Table 7.

[0315] Comparative Example 38

[0316] In this example, the amount of mercaptoethanol is adjusted to 30 μL / mL, and all other operations are consistent with those in Comparative Example 36.

[0317] The experimental results are shown in Table 7.

[0318] Comparative Example 39

[0319] In this example, the sucrose component in the cell nuclear lysate was removed, and all other operations were consistent with those in Comparative Example 36.

[0320] The experimental results are shown in Table 7.

[0321] Comparative Example 40

[0322] In this example, 10 mM EDTA-Na2 is additionally added to the cell nuclear lysate, and all other operations are consistent with comparative example 39.

[0323] The experimental results are shown in Table 7.

[0324] Comparative Example 41

[0325] In this example, the concentration of EDTA-Na2 in the cell nuclear lysate is adjusted to 5 mM, and all other operations are consistent with Comparative Example 39.

[0326] The experimental results are shown in Table 7.

[0327] Comparative Example 42

[0328] In this example, the concentration of EDTA-Na2 in the cell nuclear lysate is adjusted to 20 mM, and all other operations are consistent with those in Comparative Example 40.

[0329] The experimental results are shown in Table 7.

[0330] Comparative Example 43

[0331] In this example, 20 mM CaCl2 is added to the cell nuclear lysate. The specific formula is shown in Table 5. All other operations are consistent with Comparative Example 40.

[0332] Table 5 Nuclear lysis solution formula

[0333]

[0334] The experimental results are shown in Table 7.

[0335] Comparative Example 44

[0336] In this example, the concentration of CaCl2 in the cell nuclear lysate is adjusted to 10 mM, and all other operations are consistent with Comparative Example 43.

[0337] The experimental results are shown in Table 7.

[0338] Comparative Example 45

[0339] In this example, the concentration of CaCl2 in the cell nuclear lysate is adjusted to 30 mM, and all other operations are consistent with Comparative Example 43.

[0340] The experiment is shown in Table 7.

[0341] Comparative Example 46

[0342] The Wash Buffer used for the cell nucleus in this example is based on 0.2U / μL RNase Inhibitor and PBS, with 0.2% (v / v) TritionX-100 added additionally, and all other operations are consistent with Comparative Example 43.

[0343] The experimental results are shown in Table 7.

[0344] Comparative Example 47

[0345] In this example, the concentration of TritionX-100 in the Wash Buffer used for the cell nucleus was adjusted to 0.5% (v / v), and all other operations were consistent with those in Comparative Example 43.

[0346] The experimental results are shown in Table 7.

[0347] Comparative Example 48

[0348] In this example, the concentration of TritionX-100 in the Wash Buffer used for the cell nucleus was adjusted to 1% (v / v), and all other operations were consistent with those in Comparative Example 43.

[0349] The experimental results are shown in Table 7.

[0350] Comparative Example 49

[0351] The composition of the Wash Buffer used for the cell nucleus in this example is based on that of Comparative Example 47, with the addition of 1% (v / v) BSA. The specific formula is shown in Table 6, and all other operations are consistent with those of Comparative Example 47.

[0352] Table 6 Nucleus Wash Buffer Formula

[0353]

[0354] The experimental results are shown in Table 7.

[0355] Comparative Example 50

[0356] In this example, the concentration of BSA in the Wash Buffer used for the cell nucleus was adjusted to 0.1% (v / v), and all other operations were consistent with those in Comparative Example 49.

[0357] The experimental results are shown in Table 7.

[0358] Comparative Example 51

[0359] In this example, the concentration of BSA in the Wash Buffer used for the cell nucleus is adjusted to 5% (v / v), and all other operations are consistent with Comparative Example 49.

[0360] The experimental results are shown in Table 7.

[0361] Comparative Example 52

[0362] In this example, the pore size of the cell screen used for cell nucleus filtration is adjusted to 40 μm, and all other operations are consistent with comparative example 49.

[0363] The experimental results are shown in Table 7.

[0364] Comparative Example 53

[0365] Based on Comparative Example 52, this example adjusts the number of cell nuclei screening times to 2 times, and all other operations are consistent with Comparative Example 52.

[0366] The experimental results are shown in Table 7.

[0367] Comparative Example 54

[0368] In this example, the pore size of the cell screen used for the second screening in Comparative Example 53 is adjusted to 30 μm, and all other operations are consistent with Comparative Example 53.

[0369] The experimental results are shown in Table 7.

[0370] Comparative Example 55

[0371] In this example, the centrifugation rate in the cell nucleus washing step is adjusted to 500g, and all other operations are consistent with comparative example 54.

[0372] The experimental results are shown in Table 7.

[0373] Comparative Example 56

[0374] In this example, the centrifugation rate in the cell nucleus washing step is adjusted to 300g, and all other operations are consistent with comparative example 54.

[0375] The experimental results are shown in Table 7.

[0376] Comparative Example 57

[0377] In this example, the centrifugation rate in the cell nucleus washing step is adjusted to 100g, and all other operations are consistent with comparative example 54.

[0378] The experimental results are shown in Table 7.

[0379] Comparative Example 58

[0380] In this example, the centrifugation time in the cell nucleus washing step is adjusted to 8 minutes, and all other operations are consistent with comparative example 54.

[0381] The experimental results are shown in Table 7.

[0382] Comparative Example 59

[0383] In this example, the centrifugation time in the cell nucleus washing step is adjusted to 12 minutes, and all other operations are consistent with comparative example 54.

[0384] The experimental results are shown in Table 7.

[0385] Comparative Example 60

[0386] This example explores the applicability of the method of the present invention to specific objects. Except for changing the experimental object to a rice stem in the two-leaf, one-heart stage, the rest of the operations are consistent with those in Example 1 of the present invention.

[0387] The experimental results show that the method takes more than 8 hours to prepare rice seedling stem protoplasts, and the number of protoplasts prepared is small and the integrity is low; in addition, the number of cell nuclei prepared by this method is very small.

[0388] Comparative Example 61

[0389] This example explores the applicability of the method of the present invention to specific objects. Except for changing the experimental objects to green radish leaves, the rest of the operations are consistent with Example 1 of the present invention.

[0390] The experimental results show that the number of protoplasts and single cell nuclei of the green radish leaves prepared by this method is small, and the fragmentation rate is relatively high.

[0391] Comparative Example 62

[0392] This example explores the applicable object targeting of the method of the present invention. Except for changing the experimental object to potato tubers, the remaining operations are consistent with Example 1 of the present invention.

[0393] The experimental results show that this method takes a long time to prepare potato tuber protoplasts and has a low yield. Correspondingly, the number of single cell nuclei obtained is also very small, and the proportion of starch granules is high.

[0394] In order to develop an experimental method for simultaneously performing single-cell and single-nucleus sequencing on Arabidopsis leaf tissue, the present invention conducted a series of comparative experiments, and the results are shown in Table 7.

[0395] The present invention creatively combines plant protoplasts and plant cell nuclear sequencing in the same tissue. According to plant biological characteristics, the outermost layer of plant cells is the cell wall that plays a mechanical support role, and all substances in the cell wall are collectively referred to as protoplasts. Therefore, considering that the preparation of a single plant cell nuclear suspension requires physical or chemical means to destroy the phospholipid bilayer, dissolve the cytoplasm and cell membrane, this will damage the integrity of the protoplasts, and may result in a subsequent single cell suspension. In the preparation, a sufficient amount of complete protoplasts cannot be obtained, and the requirements for 10x Genomics cannot be met. Therefore, the present invention adopts an operating procedure of first separating protoplasts and then preparing cell nuclei in the same tissue, so as to obtain protoplasts and cell nuclear suspensions at the same time in the same tissue, ensuring that single cell and single cell nuclear sequencing can be performed simultaneously.

[0396] According to the biological characteristics of plants, the present invention will conventionally choose to prepare protoplasts first and then prepare nuclei. In the prior art, the preparation of plant protoplasts mainly uses enzymatic hydrolysis, and mechanical methods can also be used; the preparation of plant nuclei is generally extracted by physical crushing or chemical lysis, and then purified to prepare plant nuclei. According to the requirements of single-cell sequencing, the protoplast viability, total protoplast volume, protoplast fragment ratio, and protoplast agglomeration ratio are used as quality control indicators for protoplasts; according to the requirements of single-cell nuclear sequencing, the total nucleus volume, nuclear fragment ratio, and nuclear agglomeration ratio are used as quality control indicators for single-cell nuclear suspensions.

[0397] According to the whole experimental operation process, a series of comparative experiments are first conducted on the methods of preparing protoplasts and nuclei in the same tissue. In comparative example 1, mechanical crushing is used to prepare Arabidopsis leaf tissue protoplasts and single cell nucleus suspension. The results show that the number of protoplasts and nuclei prepared by mechanical grinding is very small, containing a large number of tissue fragments, and also containing organelle impurities such as chloroplasts, and the length of blade grinding time and the magnitude of force are difficult to master. Therefore, it is explained that the method of preparing Arabidopsis leaf tissue protoplasts and single cell nucleus suspension by mechanical crushing is not feasible. Subsequently, in comparative example 2, the present invention adopts the method of first removing the cell wall by enzymolysis to obtain protoplasts, and then preparing single cell nucleus suspension by mechanically crushing the cell membrane of the remaining tissue. The results show that the number of protoplasts obtained by enzymolysis has a significant increase compared with the mechanical grinding method, and the tissue fragments have a significant decrease, but the number of prepared nuclei is still small, only 4000, the proportion of nucleus fragments is as high as 50%, and the proportion of nucleus agglomeration is as high as 48%. Comparative Example 3 uses enzymatic hydrolysis to remove the cell wall to obtain protoplasts, and then adds a chemical agent to the remaining tissue to lyse the cell membrane to obtain a nuclear suspension, so as to achieve the purpose of preparing protoplasts and nuclear suspensions from the same tissue at the same time, as shown in Table 7. The results show that the quality of the protoplasts obtained by the method selected in Comparative Example 3 basically meets the requirements of single-cell transcriptome sequencing, and although the number of prepared nuclei is not large, the proportion of nuclear fragments and the proportion of nuclear agglomeration are also large, compared with Comparative Examples 1 to 2, the quantity and quality of the obtained single nuclear suspension are greatly improved.

[0398] Through the experimental verification of comparative examples 1 to 3, it is feasible to prepare protoplasts and single-cell nuclear suspension from the same Arabidopsis leaf tissue by first removing the cell wall by enzymatic hydrolysis, recovering the remaining tissue and then adding chemical reagents to lyse the cell membrane. However, simply superimposing the existing methods for preparing plant protoplasts and preparing single-cell nuclei together, the results obtained show that the two are mutually opposed, and the protoplasts and single-cell nuclear suspensions prepared in the same tissue cannot carry out single-cell and single-cell nuclear sequencing at the same time, that is, it is impossible to simultaneously prepare high-quality Arabidopsis leaf tissue protoplasts and single-cell nuclear suspensions. The reason may be that the original tissue amount distribution for preparing protoplasts and single-cell nuclear suspensions is unreasonable. During the experiment, the present invention observed that the Arabidopsis leaf tissue slices in the enzymatic hydrolysis solution were completely scattered, and some of them were translucent and colloid. Therefore, the present invention obtains as many nuclei as possible from the same sample by controlling the state of the remaining tissue after enzymatic hydrolysis, while ensuring the quantity and quality of Arabidopsis leaf tissue protoplasts.

[0399] The present invention attempts to adjust the width of the Arabidopsis leaf tissue slice after material pretreatment to control the state of the remaining tissue after enzymatic hydrolysis. In Comparative Examples 4 to 6, the widths of the initial Arabidopsis leaf tissue slices were 0.5 to 1 mm, 2 to 4 mm, and 4 to 6 mm, respectively. The protoplast viability, total protoplast volume, and total nucleus volume were used as core quality control indicators for evaluation. The results showed that when the width of the tissue slice was 0.5 to 1 mm (Comparative Example 4), the required enzymatic hydrolysis time was 30 min, and the number of protoplasts prepared was 2.6 × 10 6 When the width of the tissue slice was 2-4 mm (Comparative Example 5), the required enzymatic hydrolysis time was 50 min, and the number of protoplasts prepared was 2.1×10 4 The protoplast viability was 85%; the number of cell nuclei prepared was 1.2×10 6 When the width of the tissue slice is 4-6 mm (Comparative Example 6), the required enzymatic hydrolysis time is 80 min, and the number of protoplasts prepared is 1.9×10 5 The protoplast viability was 80%; the number of cell nuclei prepared was 1.3×10 6 indivual.

[0400] After experimental verification of comparative examples 4 to 6, when the width of Arabidopsis leaf tissue slices is 4 mm or less, as the width of the tissue slice increases, the time required for protoplast hydrolysis gradually increases, and the number of prepared nuclei increases; when the width of the tissue slice is greater than 4 mm, the time required for protoplast hydrolysis is extended, but the number of prepared nuclei is equivalent to that when the width of the tissue slice is 4 mm. Therefore, based on the experimental results and time cost, under the experimental method conditions that can meet the single-cell nuclear sequencing of Arabidopsis leaf tissue, the leaf cutting width of 2-4 mm is the preferred implementation method.

[0401] However, after adjusting the state of the remaining tissue after enzymatic hydrolysis, the prepared single-cell nuclear suspension was improved, but the number of prepared protoplasts was reduced and could not meet the requirements of single-cell sequencing. Based on this, in Example 7, the enzymatic hydrolysis time was extended to 120 min on the basis of the Arabidopsis leaf tissue cutting width of 2-4 mm. The results showed that the number of prepared protoplasts was 2.63×10 4 , there was no significant change compared with Comparative Example 5, and the number of prepared nuclei decreased slightly. In Comparative Example 8, on the basis of the cutting width of Arabidopsis leaf tissue being 2-4 mm, the concentration of cellulase R-10 in the enzymatic hydrolyzate was adjusted to 4%, and the concentration of macerate was adjusted to 0.8%. The results showed that the number of prepared protoplasts was 2.58×10 4 There was no significant change compared to Example 5, and the number of cell nuclei prepared did not change significantly.

[0402] Comparative Examples 7 to 8 show that prolonging the enzymatic hydrolysis time of leaf tissue and increasing the concentration of enzymes in the enzymatic hydrolysis solution cannot obtain protoplasts that meet the requirements of single-cell sequencing. Therefore, the present invention attempts to change the components of the enzymatic hydrolysis solution used to prepare protoplasts to explore the enzymatic hydrolysis solution formula for preparing protoplasts that meet the requirements of the machine. In Comparative Examples 9 to 15, the present invention explored the types of mixed enzymes contained in the enzymatic hydrolysis solution. The results of Comparative Example 9 show that the number of protoplasts prepared after adding 1% (m / v) pectinase is 1.6×10 6 The total number of nuclei is 2.9×10 4 The results of Example 10 showed that the number of protoplasts prepared after adding 2% (m / v) hemicellulase was 1.7×10 6 The total number of nuclei is 2.4×10 4 The number of protoplasts increased slightly, while the number of nuclei decreased. The results of Comparative Example 11 showed that the number of protoplasts prepared after adding 0.4% (m / v) lysase was 2×10 6 The total number of nuclei is 1.5×10 4The number of protoplasts increased, while the number of nuclei decreased significantly. The results of Comparative Example 12 showed that after adding 1% (m / v) xylanase, the number of protoplasts prepared was 1.9×10 6 The total number of nuclei is 8×10 4 The number of protoplasts increased, and the number of cell nuclei also increased significantly. The results of Example 13 showed that after adding 2% (m / v) snail enzyme, the number of protoplasts prepared was 1.6×10 6 The total number of nuclei is 1.2×10 4 The number of protoplasts did not change significantly, while the number of nuclei decreased significantly. The results of Comparative Example 14 showed that the number of protoplasts prepared by replacing 2% (m / v) cellulase R-10 with 2% (m / v) cellulase RS was 1.8×10 6 The total number of nuclei is 3.1×10 4 The results of Comparative Example 15 show that when the protoplast hydrolysate contains 2% (m / v) of cellulase RS, 0.4% (m / v) of macerate R-10 and 1% (m / v) of xylanase, the number of protoplasts prepared is 2.1×10 6 The total number of nuclei is 1.2×10 5 The number of protoplasts was greatly increased, and the number of cell nuclei was also significantly increased. In summary, the enzymatic solution selected by the present invention mainly contains 2% of cellulase RS, 0.4% (m / v) of macerate R-10, and xylanase.

[0403] Furthermore, the present invention determines the concentration range of the enzyme used in the enzymatic hydrolyzate by comparing Examples 16 to 18. In Comparative Example 16, when the xylanase concentration is lower than 0.5%, the number of protoplasts is 3×10 5 The total number of nuclei is 1.2×10 4 In Comparative Example 18, when the xylanase concentration was higher than 2%, 0.4% and 1%, the number of protoplasts was 3×10 6 The total number of nuclei is 0.9×10 5 The number of protoplasts increased, but the number of nuclei decreased. Considering the preparation of protoplasts and single nuclei, components, and the damage of high-concentration enzymes to protoplasts from the same sample, the concentration of xylanase in the protoplast hydrolysate should be 0.5-1% (m / v).

[0404] Considering the influence of the osmotic pressure of the cell environment on maintaining the stability of the cell membrane, the osmotic pressure of the mannitol solution is relatively high, and mannitol is replaced by sorbitol with a relatively low osmotic pressure. In comparative examples 19 to 21, the remaining tissue state after enzymolysis is good, and the experimental results show that the use of sorbitol does not affect the number of protoplasts prepared, and increases the number of cell nuclei. In addition, sorbitol is moderately priced and widely used. Taking all factors into consideration, the main buffer component for maintaining the osmotic pressure of the solution in the enzymolysis solution is determined to be 8% sorbitol.

[0405] After a series of explorations and attempts in comparative examples 4 to 21, the present invention has obtained the optimal conditions for satisfying the reasonable distribution of the original tissue amount for preparing protoplasts and single cell nucleus suspensions. That is, the protoplast hydrolysate should be: 2% cellulase RS, 0.4% (m / v) macerate R-10, 0.5-1% (m / v) xylanase, 8% (m / v) sorbitol, 20mM MES (PH=5.7), 20mM KCl, 10mM CaCl2 and 0.1% (v / v) BSA; the cutting width of Arabidopsis leaf tissue should be 2-4mm.

[0406] After the above parameters that affect the number of protoplasts and nuclei are determined, the total amount of protoplasts, viability, fragmentation rate, agglomeration ratio, the total amount of nuclei, fragmentation ratio, and agglomeration ratio are used as the main quality control indicators with reference to the 10x Genomics company standards, and are comprehensively compared and determined. The present invention then optimizes and purifies the initially obtained protoplast suspension.

[0407] The state of protoplasts is affected by temperature. Generally, at room temperature, cells maintain normal physiological metabolic activities. In comparative example 22, after the protoplasts are dissociated, the temperature is adjusted to 4 ° C during washing and purification. It is unexpectedly found that the number of protoplasts prepared is compared with room temperature (25 ° C), that is, the number of protoplasts prepared by the previous comparative example is increased. It may be that low temperature slows down cell activity and reduces its own enzymatic demise. Comparative examples 22 to 26 verify the washing conditions, i.e., centrifugal lifting speed and centrifugal time. The results show that with the decrease of centrifugal acceleration and deceleration, the integrity of the prepared protoplasts increases and the proportion of agglomeration decreases; with the increase of centrifugal time, the number of prepared protoplasts increases slightly, but the number of damage also increases. It is speculated that protoplasts are very fragile, and the acceleration and deceleration during washing and centrifugation have a certain effect on protoplasts. The centrifugal time is too long and the protoplasts are squeezed too long, causing damage. In summary, it is preferred that the protoplast preparation conditions are 4 ° C, 0 centrifugal acceleration and deceleration during centrifugal washing, and the centrifugal time is within the range of 7 to 10 min.

[0408] On the basis of the method of preparing protoplasts from plant samples, further exploration and improvement of the preparation and purification of cell nuclei are needed. In addition to the cell wall, the preparation of plant cell nuclei is also affected by plasmodesmata, intracytoplasmic organelles and secondary metabolites. The separation and purification of cell nuclei using enzymatically hydrolyzed Arabidopsis leaf tissue requires comprehensive consideration of tissue pretreatment, extraction solution selection and purification methods.

[0409] First, in terms of tissue pretreatment, an attempt was made to add a physical crushing step to the Arabidopsis leaf tissue remaining after enzymatic hydrolysis before preparing the cell nucleus suspension. In Comparative Example 27, the remaining tissue after enzymatic hydrolysis was crushed in a room temperature crusher at 1200 rpm for 30 seconds; in Comparative Example 28, the remaining tissue after enzymatic hydrolysis was crushed using liquid nitrogen. The results showed that adding a physical crushing step before chemical lysis can increase the amount of cell nuclei obtained, and the method of using liquid nitrogen crushing has a better effect.

[0410] After the tissue pretreatment experiment operation is determined, the number of cell nuclei still cannot meet the subsequent purification experiment, indicating that the existing technology is not suitable for the present invention. Further, the extraction of cell nuclei needs to be explored and improved. Ficoll 400 is a polymer of sucrose and epichlorohydrin, which is often used as a density gradient medium combined with centrifugation to remove cell debris. Polyvinylpyrrolidone (PVP), as a non-ionic polymer compound, can form a complex with phenols and alkaloids. The formation of the complex can achieve the removal of phenols and alkaloids in the sample and reduce the interference of secondary metabolites in the cell; at the same time, it can also reduce the adhesion of the cell nucleus to the endoplasmic reticulum, etc., and improve the quality of the cell nucleus. In comparative example 29, 2.5% (w / v) of Ficoll 400 in the cell nucleus lysate was replaced with PVP40. The results show that the fragment contamination of the prepared cell nucleus suspension is reduced compared with the addition of Ficoll 400. This shows that the effect achieved by adding PVP40 to the cell nucleus lysate is better than that of Ficoll 400. Furthermore, the present invention adjusts the concentration of PVP40 to 0.2% and 2% respectively, and conducts comparative examples 30 and 31 to verify the optimal concentration range. Combining the results of these three groups of comparative examples, it is concluded that the preferred concentration of PVP40 in the cell nuclear lysate is 1%.

[0411] The integrity of the biofilm is affected by the pH value of the environment. In Comparative Example 32, Tris-HCl (PH = 7.4) in the cell nucleus lysate was replaced with 10mM MOPS and 20mM sodium citrate. The experimental results show that after adding MOPS and sodium citrate, the number of prepared cell nuclei increased compared with the addition of Tris-HCl (PH = 7.4), and the proportion of cell nucleus agglomeration decreased. This shows that the solution environment provided by MOPS and sodium citrate is more conducive to the stability of cell nuclei. Therefore, MOPS and sodium citrate are more suitable than Tris-HCl (PH = 7.4). Further, the present invention explores the appropriate concentrations of MOPS and sodium citrate. In Comparative Example 33, 20mM MOPS and 30mM sodium citrate were added; in Comparative Example 34, 30mM MOPS40mM sodium citrate were added. The experimental results show that the agglomeration ratio of the single cell nucleus suspension prepared in Comparative Example 35 is lower than that of the other two groups. Therefore, preferably, the concentrations of MOPS and sodium citrate are 20 mM and 30 mM, respectively.

[0412] After the cell membrane is lysed and the cell nucleus is released, the nucleic acid molecules are easily oxidized and degraded, and a reducing agent is added to inhibit oxidative damage. In addition to the original dithiothreitol (DTT) in the cell nuclear lysate, the present invention also tried two reducing agents, mercaptoethanol and ascorbic acid, such as comparative examples 35 and 36. The experimental results show that the single cell nucleus suspension prepared using mercaptoethanol as a reducing agent is superior to the other two in quantity and quality. Furthermore, after adding comparative examples 37 to 38, it was concluded that the preferred mercaptoethanol concentration is 20 μL / mL.

[0413] Although sucrose can change the degree of nuclear precipitation and collect more nuclei in combination with gradient centrifugation, the sucrose solution with high concentration has a large viscosity and will collect more impurities and may also cause the nucleus to shrink. In comparative example 39, the sucrose component in the nuclear lysate was removed, and the results showed that the prepared nucleus was not significantly different from that with the sucrose component added. This shows that whether or not to add sucrose has no significant effect on the preparation of the nucleus.

[0414] Next, the present invention attempts to further add EDTA-Na2 as a chelating agent based on the determined cell nucleus lysate components to inhibit the enzyme activity in the solution where the cell nucleus is located; CaCl2 is added to provide a certain ionic strength to stabilize the cell nucleus state. The experimental verification of Comparative Examples 40 to 42 shows that adding 10mM EDTA-Na2 to the cell nucleus lysate can increase the number of obtained cell nuclei; the experimental verification of Comparative Examples 43 to 45 shows that adding 10mM CaCl2 to the cell nucleus lysate can improve the quality of the obtained cell nuclei.

[0415] After exploring and trying comparative examples 27 to 45, it was concluded that the preferred formula of the cell nuclear lysis solution is: 45mM MgCl2, 20mM CaCl2, 20mM 3-morpholinepropanesulfonic acid (MOPS), 30mM sodium citrate, 5% (m / v) dextran T-40 (Dextran T40), 1% (w / v) polyvinylpyrrolidone (PVP40), 0.2% (v / v) immunostaining permeabilization solution TritonX-100, 10mM EDTA-Na2, 20μL / mL of mercaptoethanol and 0.4U / μL of RNase Inhibitor.

[0416] The single cell nucleus suspension prepared in the above comparative example still has a certain degree of debris contamination, which is not conducive to single cell nucleus sequencing. The lysed cell nucleus suspension needs to be further purified later, so the present invention then discusses the influence of the Wash Buffer components used for rinsing the cell nucleus on the preparation of single cell nuclei. In comparative example 46, the Wash Buffer used for the cell nucleus tried to add 0.2% TritionX-100 on the basis of the original components. The results show that the proportion of fragments in the single cell nucleus suspension prepared by adding TritionX-100 has decreased compared with the previous one. Although TritionX-100 is a relatively mild non-ionic detergent, in addition to being used for cell membrane lysis, it also has strong foaming properties and can be used as a detergent. This shows that adding TritionX-100 to the Wash Buffer can remove some organelles such as chloroplasts and play a role in removing fragments. Furthermore, the present invention adjusts the concentration of TritionX-100 to 0.5% and 1%, respectively, and carries out comparative examples 47 and 48 to verify the optimal concentration range. Based on the results of these three groups of comparative examples, the concentration of TritionX-100 in the Wash Buffer is preferably 0.5-1%. Since the sample suspension is required to be free of metal cations in the 10x Genomics single-cell sequencing experiment, further exploratory experiments were conducted to remove metal cations from the cell nucleus suspension. In comparative example 49, an additional 1% BSA was added to the Wash Buffer used for the cell nucleus based on the determined components. The results showed that the added BSA can remove metal cations such as Mg + A type of metal cation pollution. The method of the present invention utilizes its ability to carry metal ions and adds it to the Wash Buffer used for the cell nucleus to explore its effect on the purification of single cell nucleus suspension. Furthermore, the present invention adjusts the concentration of BSA to 0.1% and 5%, respectively, and conducts comparative examples 50 and 51 to verify the optimal concentration range. Based on the results of these three groups of experiments, the preferred BSA concentration in the Wash Buffer is 0.5%.

[0417] After exploring and trying comparative examples 46 to 51, it was found that the preferred formula of Wash Buffer for cell nuclei is: 0.5-1% TritionX-100, 1% BSA, 0.2U / μL RNAse inhibitor and PBS.

[0418] After determining the formula of the cell nucleus lysate and the formula of the washing buffer, a series of exploratory verification experiments were carried out to further optimize the quality of the cell nucleus suspension. After the cells are broken, a part of the fragments and cytoplasmic components from the cell wall and cell membrane are usually filtered to remove, but at the same time, the harvest of the cell nucleus is also reduced. Based on this, the present invention explores suitable filtering conditions by changing the number of filtrations and the aperture of the cell mesh used during filtration. When the number of filtrations is once and the aperture of the cell mesh used is 70μm, the number of prepared cell nuclei is large, and accordingly, a large amount of cell fragments are also included. The number of filtrations is once, and the aperture of the cell mesh used is 40μm. The number of prepared cell nuclei is not significantly reduced compared to 70μm, and the amount of large fragments contained is reduced. The number of filtrations is twice, and the aperture of the cell mesh used is 40μm, and the number of prepared cell nuclei and the amount of fragments are reduced accordingly. The filtration times were two, and the aperture of the cell sieve used was 40 μm for the first time and 30 μm for the second time. The number of prepared cell nuclei and fragments were further reduced, but the overall quantity requirements for the machine were still met, and the purity was significantly improved. Based on the above experimental results, it was determined that the cell nuclei were first filtered through a 40 μm sieve and then through a 30 μm sieve as the optimal filtering implementation and conditions.

[0419] In addition, according to the difference between the weight of the nucleus and the weight of other fragments such as organelles, the present invention explores the appropriate centrifugal force value by changing the centrifugal force during washing centrifugation. Comparative Examples 54 to 57 correspond to centrifugal forces of 1000g, 500g, 300g and 100g respectively. The experimental results show that with the decline of centrifugal force, the proportion of collected nucleus fragments and the proportion of agglomeration also decrease accordingly, and accordingly, the number of collected nuclei also decreases accordingly. Although high-speed centrifugation can collect more nuclei, it is easy to rupture the nuclei and increase the contamination of other non-nuclear substances in the nucleus suspension. For example, when the centrifugal force is 1000g, the proportion of nucleus fragments reaches 16%, and there is a greater risk of failure in subsequent machine operation. Combining several groups of experimental results, when the number is guaranteed to meet the requirements of nucleus sequencing, the fragmentation rate and agglomeration rate of the obtained nucleus suspension are the lowest when the centrifugal rate is 300g. Furthermore, the present invention explored the appropriate centrifugation time for the cell nucleus washing step. The experimental results of comparative examples 54, 58 and 59 showed that when the centrifugation time was between 6 and 8 minutes, the collected single cell nucleus suspension met the requirements of single cell nucleus sequencing. When the time exceeded 8 minutes, the proportion of prepared cell nuclei agglomeration was higher and the amount of fragments also increased accordingly.

[0420] To illustrate that the method of the present invention is mainly applicable to Arabidopsis leaf tissue, the present invention conducted experiments of comparative examples 60 to 62, respectively using rice seedlings with two leaves and one heart period stems, green radish leaves and potato tubers as experimental objects, and simultaneously preparing protoplasts and nuclei for the same tissue. The experimental results show that it takes a long time to prepare rice protoplasts using the method provided by the present invention, and there is basically no remaining tissue material after enzymatic hydrolysis that can be used to prepare nuclei; the number of protoplasts and nuclei prepared from green radish leaves using the method provided by the present invention is small, and there are many nuclear fragments; it takes a long time to prepare potato tuber protoplasts using the method provided by the present invention, and the number is small, containing a large number of impurities such as starch granules, and almost no nuclei are prepared.

[0421] In summary, in the extraction method for simultaneously preparing Arabidopsis leaf tissue protoplasts and single cell nuclei described in the present invention, there is a one-to-one correspondence between Arabidopsis leaves, single cell sequencing and single cell nucleus sequencing results. Through the experimental verification of comparative examples 1 to 3, it is concluded that the method for simultaneously preparing protoplasts and single cell nucleus suspensions from the same Arabidopsis leaf tissue is to obtain protoplasts by enzymatic hydrolysis, and then retain the tissue to lyse and prepare the nuclei. Through the experimental verification of comparative examples 4 to 21, it is concluded that the preferred enzymatic hydrolyzate and WI buffer formulas, and the preferred tissue slice conditions. Through the experimental verification of comparative examples 22 to 26, it is concluded that the preferred experimental parameters for preparing protoplasts are obtained. Through the experimental verification of comparative examples 27 to 28, it is concluded that the preferred method for preparing nucleus tissue pretreatment is obtained. Through the experimental verification of comparative examples 29 to 51, the preferred formula of nucleus lysate and Wash Buffer is obtained. Through the experimental verification of comparative examples 51 to 59, the operating details and experimental parameters of each experimental step for preparing Arabidopsis leaf tissue nuclei are clarified; through comparative examples 60 to 62, it is concluded that the compatibility of the method proposed by the present invention with Arabidopsis leaf tissue. The present invention describes a method for preparing protoplasts and single cell nuclei from the same Arabidopsis leaf tissue, which can effectively prepare the protoplast suspension and single cell nucleus suspension required for single cell and single cell nucleus sequencing experiments on the 10x Genomics platform.

[0422] Table 7

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the present invention. Modifications or equivalent replacements of the technical solution of the present invention are all within the essence and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously preparing protoplasts and single cell nuclei from Arabidopsis leaf tissue, characterized in that: The method comprises the following steps: (1) Cut Arabidopsis leaves, rinse them and cut them evenly into tissue slices, put them into a culture dish, add WI buffer to grind them, and then transfer them to a culture dish containing enzymatic solution, place them on a constant temperature mixer to digest in the dark, and obtain digestion solution and digested Arabidopsis leaf tissue; the width of the cut tissue slices is 2-4 mm; the formula of the enzymatic solution is: 2% m / v cellulase RS, 0.4% m / v macerate R-10, 0.5-1% m / v xylanase, 8% m / v sorbitol, 20mM MES with a pH of 5.7, 20mM KCl, 10mM CaCl2 and 0.1% v / v BSA; the formula of the WI buffer is: 0.5M mannitol, 4mM MES with a pH of 5.7 and 20mM KCl; (2) Slowly sieve the digestion solution to obtain the sieved supernatant and the remaining Arabidopsis leaf tissue after digestion. Rinse the remaining tissue with WI buffer, filter the rinse solution into the sieved supernatant to obtain the filtrate, which contains protoplasts, and retain the remaining Arabidopsis leaf tissue; The formula of the WI buffer is: 0.5M mannitol, 4mM MES with pH=5.7 and 20mM KCl; (3) The filtrate is centrifuged for the first time, the supernatant is discarded to obtain a precipitate, the precipitate is resuspended in WI buffer and then centrifuged for a second time, the supernatant is discarded to obtain a precipitate, the precipitate is resuspended in WI buffer to obtain a protoplast suspension, and the protoplast suspension is counted under a microscope; the temperature during the first and second centrifugations is 4°C, 100×g, the centrifugal lifting speed is 0, and the time is 7 to 10 min; the formula of the WI buffer is: 0.5 M mannitol, 4 mM MES with a pH of 5.7, and 20 mM KCl; (4) Transfer the Arabidopsis leaf tissue remaining after digestion retained in step (2) into an EP tube, add steel beads into the tube, crush with liquid nitrogen, and then add lysis buffer for lysis; the formula of the lysis buffer is: 45mM MgCl2, 20mM CaCl2, 20mM 3-morpholinepropanesulfonic acid, 30mM sodium citrate, 5% m / v dextran T-40, 1% w / v polyvinylpyrrolidone PVP40, 0.2% v / v immunostaining permeabilization solution TritonX-100, 10mM EDTA-Na2, 20μL / mL mercaptoethanol and 0.4U / μL RNase Inhibitor; (5) Adding a washing buffer to the incubated lysate and sieving it to obtain a sieved supernatant; the formula of the washing buffer is: 0.5-1% TritionX-100, 1% BSA, 0.2U / μL RNAse inhibitor and PBS; the sieving times are 2 times, the pore size of the cell sieve used in the first time is 40 μm, and the pore size of the cell sieve used in the second time is 30 μm; (6) The sieved supernatant was centrifuged twice, the supernatant was discarded and the precipitate was retained, and the single cell nucleus suspension was obtained by resuspending in the washing buffer, and then counted under a microscope; the first centrifugation was performed at 300g for 6-8min, and the adapted temperature was 4°C; the second centrifugation was performed at 300g for 8-10min, and the adapted temperature was 4°C; the formula of the washing buffer was: 0.5-1% TritionX-100, 1% BSA, 0.2U / μL RNAse inhibitor and PBS.

2. The method according to claim 1, characterized in that The protoplasts obtained by the method have high purity and low fragmentation rate, and the number of protoplasts reaches 2.8×10 6 ~2.83×10 6 The survival rate of protoplasts was 93% to 94%, the clumping rate was 3%, and the fragmentation rate was 2%. The number of nuclei obtained by the method was 1.6×10 6 ~1.65×10 6 The proportion of nuclear agglomeration was 3%, and the proportion of nuclear fragments was 5%~6%.

3. A method for simultaneous protoplast and single cell nucleus sequencing of Arabidopsis leaf tissue, characterized in that: The method uses the protoplasts and single cell nuclei prepared by the method of claim 1 for sequencing.

4. A kit, characterized in that: The kit is used for preparing a protoplast suspension and a single cell nucleus suspension in the same Arabidopsis leaf tissue and sequencing them simultaneously; the kit comprises ① a protoplast hydrolysate, the formula of which is: 2% m / v cellulase RS, 0.4% m / v maceration enzyme R-10, 0.5-1% m / v xylanase, 8% m / v sorbitol, 20mM MES with a pH value of 5.7, 20mM KCl, 10mM CaCl2 and 0.1% v / v BSA; ② a WI buffer, the formula of which is: 0.5M mannitol, 4mM MES with a pH value of 5.7 and 20mM KCl; ③ a cell nucleus lysis solution, the formula of which is: 45mM MgCl2, 20mM CaCl2, 20mM 3-morpholinepropanesulfonic acid, 30mM sodium citrate, 5% m / v ④ Dextran T-40, 1% w / v polyvinylpyrrolidone PVP40, 0.2% v / v immunostaining permeabilization solution TritonX-100, 10mM EDTA-Na2, 20μL / mL mercaptoethanol and 0.4 U / μL RNase Inhibitor; ④ Cleaning buffer, whose formula is: 0.5~1% TritionX-100, 1% BSA, 0.2U / μL RNAse inhibitor and PBS.

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