A plant nucleus preparation and suspension optimization method suitable for single-cell sequencing of kiwifruit root tissue

Abstract

The present invention discloses a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing. The nuclei are prepared with high quality through an original lysate formula and a nucleus suspension optimization method. Compared with the lysate formula of 6 to 7 components in the prior art, the lysate formula of the present invention is significantly reduced; the experimental process can complete the preparation of nuclei within 30 minutes. The method of the present invention avoids steps such as sucrose deposition, saves reagents and consumables, reduces costs, and greatly shortens time, improves efficiency, and facilitates the development of experiments with a large number of samples. The present invention also discloses a KR lysate, a reagent / kit, and its use in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing plant cell nuclei, and specifically relates to a method for preparing cell nuclei of kiwifruit root tissue and an application and use method thereof in plant single-cell sequencing. Background Art

[0002] Single-cell sequencing technology measures the transcriptome of a single cell and uses "single-cell resolution" to analyze the molecular mechanisms behind life phenomena. It is a cutting-edge technical means for life science research and has been widely used. However, due to the presence of cell walls, single-cell sequencing of plant tissues is "difficult". Plant cells must first be dissociated into protoplasts before single-cell sequencing can be performed. The main components of the cell wall are polysaccharides such as cellulose. These macromolecules have variable structures and stable chemical properties and are one of the most difficult substances to degrade. Therefore, it is currently difficult to prepare protoplasts that meet the requirements of single-cell sequencing from most plant tissues, which greatly limits plant single-cell sequencing research. In addition, the preparation of protoplasts causes very large changes to plant cells, which will induce transcriptional changes, resulting in a serious reduction in the reliability of sequencing results, and the data cannot reflect real biological changes.

[0003] In order to circumvent the difficulties in separating cells from tissues and data distortion caused by the dissociation process, animal tissues will use single-cell nuclear sequencing to conduct experiments, which has achieved very good results. Therefore, borrowing from the methods used in animal tissues and conducting plant single-cell sequencing by preparing plant cell nuclei is an effective means to overcome current technical difficulties. However, the current methods for preparing cell nuclei from plant tissues are all applied to related research on Arabidopsis thaliana, and are not suitable for kiwifruit root tissue research. After preparing cell nuclei from kiwifruit root tissues, indicators such as the number of cell nuclei and the proportion of cell fragments cannot meet the requirements of single-cell sequencing. It is urgent to develop a new method for preparing cell nuclei suitable for single-cell sequencing research of kiwifruit root tissue plants. Summary of the invention

[0004] Although Arabidopsis has mature cell nucleus preparation technology solutions, such as the experimental steps described in "Identification of OpenChromatin Regions in Plant Genomes Using ATAC-Seq" and "Isolation of Plant RootNuclei for Single Cell RNA Sequencing", the former is mainly used in Arabidopsis genome sequencing research, and the latter is mainly used in transcriptome sequencing research. The two methods can prepare cell nucleus suspensions of Arabidopsis leaf and root tissues, respectively. Kiwifruit is a large deciduous vine with very low tissue similarity to Arabidopsis, with large differences in cell wall composition, and due to different growth environments, the composition of tissues and cells is also very different. The method of preparing cell nuclei using Arabidopsis cannot prepare kiwifruit root tissue cell nucleus suspensions that meet the requirements of single-cell sequencing.

[0005] In view of the gaps and shortcomings in the prior art, the present invention, based on a large amount of in-depth research and experiments, provides a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, as well as a corresponding lysate formula and a method for optimizing a cell nucleus suspension (wherein the cell nucleus suspension optimization method described in the present invention mainly optimizes the removal of calcium oxalate crystals deposited in kiwifruit root tissue / cells, the removal of mRNA released into the suspension during the extraction of kiwifruit root tissue, and the removal of tissue fragments in the cell nucleus suspension).

[0006] The method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing provided by the present invention comprises the following specific steps:

[0007] 1) Prepare KR lysis buffer: First prepare KR lysis buffer and pre-cool the prepared solution.

[0008] 2) Sample crushing: transfer freshly collected or frozen kiwifruit root tissue samples into a centrifuge tube, add pre-cooled KR lysis solution and steel beads, and crush the tissue on a crusher according to the instrument operating instructions.

[0009] 3) Lyse cells: After disruption, place the centrifuge tube on ice and incubate for lysis.

[0010] 4) Screen filtration: Transfer the tissue lysate after lysis in step 3) into a centrifuge tube and add pre-cooled PBS. Let stand on ice for 1 minute. Aspirate the tissue lysate and add it to the filter for filtration.

[0011] 5) Screen filtration: Use a screen to filter the filtrate in step 4) again.

[0012] 6) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 5), discard the supernatant, and resuspend the precipitate in PBS.

[0013] 7) Filtration: Add all the precipitate resuspension in step 6) into the separation column for filtration.

[0014] 8) Collecting the precipitate by centrifugation: Centrifuge the filtrate in step 7), discard the supernatant, and resuspend the precipitate in 100 μL PBS.

[0015] In step (1), the KR lysate contains the following components (final concentration):

[0016] 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2 to 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613); preferably, 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 25 mM sodium citrate, 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613).

[0017] In step (1), L-tartaric acid is commonly used as an acidulant for beverages and other foods. In the present invention, it serves to neutralize the cell contents released after tissue and cell disruption, stabilize the pH of the lysate, and maintain the integrity of the cell nucleus. At the same time, L-tartaric acid acts as a reducing agent in the lysate to maintain the activity of the RNase inhibitor.

[0018] In step (1), saponin mainly functions as a pharmaceutical raw material, and is also used as a preservative material and an emulsifier component. In the present invention, saponin, as a component of a lysing solution, plays a role in lysing cell membranes and releasing cell nuclei.

[0019] In step (1), when preparing the KR lysate, 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) needs to be added first, the solution is thoroughly mixed and placed on ice for precooling before adding 0.2 to 0.4 U / mL RNase inhibitor. The purpose is that 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) can maintain the activity of 0.2 to 0.4 U / mL RNase inhibitor.

[0020] In step (1), the precooling temperature is 0-5°C; preferably, it is precooled in a 4°C refrigerator.

[0021] In step (2), the centrifuge tube is 2.0mL MaxyClear Snaplock Microcentrifuge Tube, RNase- / DNase-free and nonpyrogenic (Axygen, MCT-200-C).

[0022] In step (2), the disruptor is commonly used in nucleic acid extraction. In the present invention, its function is to fully destroy plant tissues through steel balls and assist KR lysis solution in completing the preparation of crude cell nucleus suspension.

[0023] In step (2), the diameter of the steel ball is 5 mm.

[0024] In step (2), the disruptor is a Wanbo Bio high-throughput tissue disruptor (onebio-48p).

[0025] In step (2), the crushing conditions are 1200-1300 rpm, 170-180 s; preferably, 1200 rpm, 180 s.

[0026] In step (2), the kiwifruit root may refer to the root tip tissue of any variety of kiwifruit, including the meristem, elongation and maturation zones.

[0027] In step (2), the fresh weight of the kiwifruit root tissue is 50-100 mg; preferably, 100 mg.

[0028] In step (3), the purpose of the incubation is to use KR lysis solution to remove the cell membrane and other structures outside the cell nucleus at a temperature that maintains the integrity of the messenger RNA (mRNA) in the cell nucleus.

[0029] In step (3), the incubation time is 7 to 8 minutes; preferably, 7 minutes.

[0030] In step (4), the filter is FALCON 40 μm Cell Strainer (FALCON, 352340) with a pore size of 40 μm.

[0031] In step (4), the PBS is Gibco PBS pH 7.4 (1×) (Gibco, 10010-031).

[0032] In step (4), the centrifuge tube is a Corning Centristar 15 mL centrifuge tube (Corning, 430790).

[0033] In step (4), the filtration is performed at 0-5°C throughout the entire process; preferably, the filtration is performed on ice.

[0034] The purpose of standing on ice for 1 minute in step (4) is to allow large tissue blocks to settle to the bottom of the centrifuge tube. If the standing time is too short, the tissue blocks will not settle completely, causing the screen to be blocked during filtration, resulting in loss of cell nuclei; if the standing time is too long, there is a risk of mRNA degradation, which will directly lead to the failure of the experiment. The tissue block sedimentation time of 1 minute can ensure complete sedimentation without the risk of mRNA degradation.

[0035] In step (5), the filter is FALCON 40 μm Cell Strainer (FALCON, 352340) with a pore size of 40 μm.

[0036] In step (6), the centrifugation temperature is 4-6°C; preferably, 4°C.

[0037] In step (6), the centrifugation time is 10-15 minutes; preferably, 10 minutes.

[0038] In step (6), the centrifugal force of the centrifugation is 300-500×g, preferably 300×g.

[0039] In step (6), the PBS is pre-cooled in a 4°C refrigerator for 30 minutes in advance.

[0040] In step (7), the separation column is Miltenyi MS Column (Miltenyi, 130-042-201).

[0041] In step (7), the sorting column is usually used in a magnetic field to sort specific cell types of animals by binding antibodies with magnetic beads to specific antigens on the surface of the target cell membrane. In the present invention, in a non-magnetic field, the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized, so that small cell nuclei can pass through the sorting column along with the liquid flow, while the needle-shaped calcium oxalate crystals will be retained in the sorting column and cannot pass through along with the liquid flow, thereby achieving the purpose of purifying cell nuclei.

[0042] In step (7), the filtration is performed at 0-5°C throughout the entire process; preferably, the filtration is performed on ice.

[0043] In step (8), the centrifugation temperature is 4-6°C; preferably, 4°C.

[0044] In step (8), the centrifugation time is 10-15 minutes; preferably, 10 minutes.

[0045] In step (8), the centrifugal force of the centrifugation is 300-500×g; preferably, 300×g.

[0046] In step (8), the PBS is pre-cooled on ice.

[0047] The present invention also provides kiwifruit root tissue cell nuclei suitable for single-cell sequencing obtained by the above method.

[0048] The present invention also provides the application of the kiwifruit root tissue cell nucleus in single-cell sequencing.

[0049] In a specific embodiment, the specific steps of the method of the present invention are:

[0050] 1) Prepare KR lysis buffer.

[0051] Prepare the lysis buffer according to the following ingredients:

[0052] 2%~3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15~25mM sodium citrate, 0.2~0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2~0.4U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613).

[0053] Place the prepared lysate on ice for pre-cooling.

[0054] 2) Crushing samples.

[0055] Transfer freshly collected or frozen kiwifruit root tissue samples into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200-1300 rpm for 170-180 seconds. Start the instrument to crush the tissue.

[0056] 3) Lyse cells.

[0057] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0058] 4) Filter.

[0059] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0060] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0061] 5) Filter.

[0062] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0063] 6) Collect the precipitate by centrifugation.

[0064] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0065] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0066] 7) Filter.

[0067] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0068] 8) Collect the precipitate by centrifugation.

[0069] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0070] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0071] 9) Microscopic examination and counting.

[0072] Take 5 μL of the resuspension in step (8) and mix it with DAPI staining solution in a ratio of 1:1. Use a hemocytometer to count the total number and concentration of cell nuclei.

[0073] 10) Single-cell sequencing on the machine.

[0074] According to the instructions of the 10x Genomics kit, single-cell sequencing was performed on the machine.

[0075] The present invention also provides a KR lysate, which comprises the following components (final concentration): 2% to 3% (v / v) L-tartaric acid, 15 to 25 mM sodium citrate, 0.2 to 0.4% (m / v) saponin, and 0.2 to 0.4 U / mL recombinant RNase inhibitor.

[0076] The present invention also provides a reagent / kit, characterized in that it comprises the KR lysate as described above.

[0077] The present invention also provides the use of the KR lysate or the reagent / kit in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, kiwifruit root tissue nuclei transcriptome sequencing, and kiwifruit root tissue nuclei ATAC sequencing research.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] First, it fills the current technical gap: the lack of a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing experiments has seriously hindered the application of single-cell sequencing technology in kiwifruit root tissue research. The technical solution provided by the present invention solves this problem and will effectively promote the development and widespread application of kiwifruit root tissue single-cell sequencing research.

[0080] Second, the quality of kiwifruit root nucleus suspension is improved. Kiwifruit root tissue contains a large amount of impurities and polysaccharides. After lysis, the suspension contains a large amount of debris, intracellular crystals and other impurities. Its quality is completely unable to meet the requirements of 10x Genomics, which seriously hinders the research progress of kiwifruit root tissue. However, the quality of the nuclei prepared according to the technical solution of the present invention is very high (the sample quality is from 3×10 5 The fragmentation rate is greater than 70%, which is increased to 2.26×10 6 ~4.12×10 6 The fragmentation rate is 3-5%. ), cell fragments, intracellular crystals and other impurities are basically completely removed, and subsequent single-cell experiments can be carried out smoothly.

[0081] Third, it is low-cost and time-saving. The technical solution provided by the present invention uses only 4 lysate components, which is significantly reduced compared with 6 to 7 components in the prior art; the experimental process can complete the preparation of cell nuclei within 30 minutes. It avoids steps such as sucrose deposition, saves reagents and consumables, reduces costs, and greatly shortens time, improves efficiency, and facilitates the development of large-scale sample experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 This is a microscopic examination of a cell nucleus. The figure shows a cell nucleus prepared by the technical method of the present invention. It can be seen in the figure that impurities such as cell debris and calcium oxalate crystals are completely removed, and only a very small proportion of impurities smaller than the cell nucleus exist.

[0083] Figure 2 It is the cDNA quality inspection result of single cell transcriptome sequencing of Examples 1 to 4 of the present invention (Agilent 4150 Bioanalyzer).

[0084] Figure 3 This is a graphic representation of needle-shaped calcium oxalate crystals in tissue. DETAILED DESCRIPTION

[0085] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0086] Example 1

[0087] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0088] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0089] 2) Crushing samples.

[0090] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 170 seconds. Start the instrument to crush the tissue.

[0091] 3) Lyse cells.

[0092] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0093] 4) Filter.

[0094] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0095] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0096] Note that all operations should be performed on ice.

[0097] 5) Filter.

[0098] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0099] Note that all operations should be performed on ice.

[0100] 6) Collect the precipitate by centrifugation.

[0101] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0102] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0103] 7) Filter.

[0104] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0105] 8) Collect the precipitate by centrifugation.

[0106] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0107] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0108] 9) Washing the precipitate

[0109] Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0110] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0111] 10) Quality inspection, microscopic examination and counting.

[0112] The supernatant was aspirated and the RNA concentration in the suspension was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0113] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (9) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0114] 11) Single-cell sequencing is carried out on the machine.

[0115] Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3' Reagent Kit v3.1, CG000204 REV C.

[0116] Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1 ), cDNA length is normal ( Figure 2 ). The library construction results showed that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples was effective.

[0117] Example 2

[0118] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0119] 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 25 mM sodium citrate, 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0120] 2) Crushing samples.

[0121] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1300 rpm for 180 seconds. Start the instrument to crush the tissue.

[0122] 3) Lyse cells.

[0123] After the instrument stops completely, take out the centrifuge tube and put it in ice for incubation for 8 minutes.

[0124] 4) Filter.

[0125] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0126] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0127] Note that all operations should be performed on ice.

[0128] 5) Filter.

[0129] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0130] Note that all operations should be performed on ice.

[0131] 6) Collect the precipitate by centrifugation.

[0132] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0133] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0134] 7) Filter.

[0135] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0136] 8) Collect the precipitate by centrifugation.

[0137] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0138] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0139] 9) Washing the precipitate

[0140] Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0141] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0142] 10) Quality inspection, microscopic examination and counting.

[0143] The supernatant was aspirated and the RNA concentration in the suspension was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0144] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (9) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0145] 11) Single-cell sequencing is carried out on the machine.

[0146] Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3'Reagent Kitv3.1, CG000204REV C.

[0147] Results and analysis: The results are shown in Table 1; the RNA content in the suspension was extremely low; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all met the requirements of 10x Genomics for single-cell sequencing, and the cDNA length was normal. The library construction results showed that the single-cell transcriptome sequencing of the cell nucleus suspension extracted from the frozen sample was effective ( Figure 2 ).

[0148] Example 3

[0149] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0150] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0151] 2) Crushing samples.

[0152] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 170 seconds. Start the instrument to crush the tissue.

[0153] 3) Lyse cells.

[0154] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0155] 4) Filter.

[0156] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0157] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0158] Note that all operations should be performed on ice.

[0159] 5) Filter.

[0160] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0161] Note that all operations should be performed on ice.

[0162] 6) Collect the precipitate by centrifugation.

[0163] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0164] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0165] 7) Filter.

[0166] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0167] 8) Collect the precipitate by centrifugation.

[0168] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0169] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0170] 9) Washing the precipitate

[0171] Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0172] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0173] 10) Quality inspection, microscopic examination and counting.

[0174] The supernatant was aspirated and the RNA concentration in the suspension was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0175] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (9) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0176] 11) Single-cell sequencing is carried out on the machine.

[0177] Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3'Reagent Kitv3.1, CG000204REV C.

[0178] Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1 ), cDNA length is normal ( Figure 2 ). The library construction results showed that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples was effective.

[0179] Example 4

[0180] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0181] 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 25 mM sodium citrate, 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0182] 2) Crushing samples.

[0183] Transfer 100 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1300 rpm for 180 seconds. Start the instrument to crush the tissue.

[0184] 3) Lyse cells.

[0185] After the instrument stops completely, take out the centrifuge tube and put it in ice for incubation for 8 minutes.

[0186] 4) Filter.

[0187] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0188] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0189] Note that all operations should be performed on ice.

[0190] 5) Filter.

[0191] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0192] Note that all operations should be performed on ice.

[0193] 6) Collect the precipitate by centrifugation.

[0194] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0195] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0196] 7) Filter.

[0197] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0198] 8) Collect the precipitate by centrifugation.

[0199] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0200] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0201] 9) Washing the precipitate

[0202] Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C.

[0203] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0204] 10) Quality inspection, microscopic examination and counting.

[0205] The supernatant was aspirated and the RNA concentration in the suspension was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0206] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (9) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0207] 11) Single-cell sequencing is carried out on the machine.

[0208] Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3' Reagent Kit v3.1, CG000204 REV C.

[0209] Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1 ), cDNA length is normal ( Figure 2 ). The library construction results showed that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples was effective.

[0210] Comparative Example 1

[0211] In this example, Arabidopsis leaf nuclei were prepared according to "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq".

[0212] 1) Prepare lysis buffer.

[0213] Prepare the nuclear purification buffer according to the following ingredients:

[0214] 20mM MOPS pH 7, 40mM NaCl, 90mM KCl, 2mM EDTA, 0.5mM EGTA, 0.5mMspermidine, 0.2mM spermine, and 1×Roche Complete protease inhibitors.

[0215] Prepare the cell nuclear extract 1 according to the following ingredients:

[0216] 0.25M Sucrose, 10mM Tris–HCl pH 8, 10mM MgCl2, 1% Triton X-100, and 1×Roche Complete Protease Inhibitors.

[0217] Prepare the nuclear extract 2 according to the following ingredients:

[0218] 1.7M Sucrose, 10mM Tris–HCl pH 8, 2mM MgCl2, and 0.15% Triton X-100, 1×Roche Complete Protease Inhibitors.

[0219] Pre-cool the prepared solution on ice.

[0220] 2) Crushing samples.

[0221] Transfer 50 mg of Arabidopsis leaf tissue sample into a mortar, add a certain amount of liquid nitrogen and grind until the tissue is completely powdered. During this period, be sure to add liquid nitrogen to keep the temperature low.

[0222] 3) Lyse cells.

[0223] Transfer the ground powder into a new mortar containing 10 mL of ice-cold nuclear purification buffer. Use a pestle to stir and resuspend the powder.

[0224] 4) Filter.

[0225] Use a pipette to gradually aspirate the tissue lysate in step (3), filter it through a 70 μm filter, and collect the filtrate into a new 15 mL centrifuge tube. Note that the centrifuge tube should be placed in ice.

[0226] 5) Collect the precipitate by centrifugation.

[0227] Centrifuge the centrifuge tube in step (4) at 1200×g and 4°C for 10 minutes.

[0228] Carefully remove the centrifuge tube and discard the supernatant.

[0229] 6) Collect the precipitate by centrifugation.

[0230] Resuspend the nuclear pellet in step (5) using 1 mL of pre-cooled nuclear extraction solution 1.

[0231] Aspirate the resuspension and add it to a new 1.5 mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 × g and 4°C for 10 minutes.

[0232] Carefully remove the centrifuge tube and discard the supernatant.

[0233] 7) Collect the precipitate by centrifugation.

[0234] Add 300 μL of Nuclear Extraction Solution 2 to a new 1.5 mL centrifuge tube.

[0235] Resuspend the nuclear pellet in step (6) using 300 μL of pre-cooled nuclear extract solution 2. Carefully add the resuspended solution to the centrifuge tube containing nuclear extract solution 2 to separate the solutions.

[0236] Place the centrifuge tube in a centrifuge and centrifuge at 16,000 × g and 4°C for 10 minutes.

[0237] Carefully remove the centrifuge tube and discard the supernatant.

[0238] Resuspend the pellet in 1 mL of pre-chilled Nuclei Purification Buffer.

[0239] 8) Microscopic examination and counting.

[0240] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the resuspension in step (8), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0241] 9) Results and analysis

[0242] The results showed that the number of cell nuclei was 2×10 6 The fragment impurities accounted for 28%, which met the minimum sample requirements for single-cell transcriptome sequencing.

[0243] Comparative Example 2

[0244] This example is based on "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" to prepare kiwifruit leaf tissue nuclei. The remaining steps are the same as those in Comparative Example 1.

[0245] The results are shown in Table 2.

[0246] Comparative Example 3

[0247] This example is based on "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" to prepare kiwifruit root tissue nuclei. The remaining steps are the same as those in Comparative Example 1.

[0248] The results are shown in Table 2.

[0249] Comparative Example 4

[0250] This example is based on the plant tissue nuclear extraction kit (CelLytic TM PN isolation / extraction kit (Sigma, number CELLYTPN1-1KT) was used to prepare Arabidopsis root tissue nuclei.

[0251] 1) Prepare lysis buffer

[0252] Pour 500 μL of NIB Lysis Buffer into a new, sterile, pre-chilled 60 mm culture dish.

[0253] 2) Draw materials

[0254] The roots of 7-day-old Arabidopsis plants were cut with a double-edged razor blade and fresh roots were collected.

[0255] 3) Transfer materials

[0256] Use forceps to transfer fresh root samples to a 60 mm Petri dish and immerse the entire root in NIB lysis solution.

[0257] 4) Tissue mincing

[0258] The obtained root samples were minced with a double-sided blade for about 5 minutes.

[0259] 5) Tissue lysis

[0260] Incubate at 4°C with gentle horizontal shaking for 15 minutes to allow lysis.

[0261] 6) Filter

[0262] Under low temperature, first use 500 μL NIB to wet the 40 μm filter, then use a disposable large-bore pipette tip to absorb the nuclear suspension after root lysis, filter it slowly, and then use 500 μL NIB to rinse the filter and collect the remaining cell nuclei.

[0263] 7) Screen filtration

[0264] The filtered nuclear suspension was then filtered again using a 30 μm filter in the same manner as step 7, and the filter was then rinsed with 500 μL of NIB to collect the remaining nuclei and remove the debris.

[0265] 8) Microscopic examination and counting

[0266] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the resuspension in step (8), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0267] The results are shown in Table 2

[0268] Comparative Example 5

[0269] This example is based on the plant tissue nuclear extraction kit (CelLytic TM PN separation / extraction kit, sigma, numbered CELLYTPN1-1KT) was used to prepare the cell nuclei of kiwifruit leaf tissue. The remaining operation steps were the same as those in Comparative Example 4.

[0270] The results are shown in Table 2.

[0271] Comparative Example 6

[0272] This example is based on the plant tissue nuclear extraction kit (CelLytic TM PN separation / extraction kit, Sigma, numbered CELLYTPN1-1KT) was used to prepare the nuclei of kiwifruit root tissue. The remaining steps were the same as those in Comparative Example 4.

[0273] The results are shown in Table 2.

[0274] Comparative Example 7

[0275] In this example, ST1 buffer was used to prepare kiwifruit root tissue cell nuclei.

[0276] 1) Prepare ST1 buffer. Prepare the buffer according to the following ingredients:

[0277] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023). Pre-cool the prepared buffer on ice.

[0278] 2) Crushing samples.

[0279] Transfer 50 mg of kiwifruit root tissue sample into a mortar, add a certain amount of liquid nitrogen and grind until the tissue is completely powdered. During this period, be sure to add liquid nitrogen to keep the temperature low.

[0280] 3) Lyse cells.

[0281] Transfer the ground powder into a new 15 mL centrifuge tube containing 10 mL of ice-cold buffer. Invert 15 times to resuspend.

[0282] 4) Microscopic examination and counting.

[0283] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the cell nucleus suspension in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0284] 5) Parameter measurement.

[0285] The pH of the buffer solution in step (3) was measured using a pH meter (METTLER TOLEDO FE28-CN, 30595013).

[0286] The results are shown in Table 2.

[0287] Comparative Example 8

[0288] In this example, ST2 buffer containing 15 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining operating steps were the same as those in Comparative Example 7.

[0289] The results are shown in Table 2.

[0290] Comparative Example 9

[0291] In this example, ST3 buffer containing 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) and 20 mM Tris-HCl (pH=7.5) was used to prepare kiwifruit root tissue nuclei. The remaining operating steps were the same as those in Comparative Example 7.

[0292] Comparative Example 10

[0293] In this example, ST4 buffer with the components of 15 mM sodium citrate and 20 mM MOPS (3-(N-morpholine)propanesulfonic acid, Sigma-Aldrich, 1132-61-2) was used to prepare kiwifruit root tissue nuclei, and the remaining operating steps were the same as those in Comparative Example 7.

[0294] The results are shown in Table 2.

[0295] Comparative Example 11

[0296] In this example, ST buffer containing 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15 mM sodium citrate was used to prepare kiwifruit root tissue nuclei. The remaining operating steps were the same as those in Comparative Example 7.

[0297] The results are shown in Table 2.

[0298] Comparative Example 12

[0299] In this example, ST5 buffer containing 1.5% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) and 10 mM sodium citrate was used to prepare kiwifruit root tissue nuclei. The remaining operations were the same as those in Comparative Example 7.

[0300] The results are shown in Table 2.

[0301] Comparative Example 13

[0302] In this example, ST6 buffer with the components of 4% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) and 30 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining operations were the same as those in Comparative Example 7.

[0303] The results are shown in Table 2.

[0304] Comparative Example 14

[0305] This example uses KR1 lysis buffer to prepare kiwifruit root tissue nuclei.

[0306] 1) Prepare KR1 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0307] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, 0.2% (m / v) SDS (sodium dodecyl sulfate, Sigma, 151-21-3). The prepared lysate was placed on ice for precooling.

[0308] 2) Crushing samples.

[0309] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and chop the kiwifruit root into pieces using sterilized scissors in the lysis solution.

[0310] 3) Lyse cells.

[0311] Place the centrifuge tube in ice and incubate for 8 minutes.

[0312] 4) Microscopic examination and counting.

[0313] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0314] The results are shown in Table 2.

[0315] Comparative Example 15

[0316] In this example, kiwifruit root tissue nuclei were prepared using KR2 lysate consisting of 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m / v) sodium deoxycholate (Sigma, 302-95-4). The remaining steps were the same as those in Comparative Example 14.

[0317] The results are shown in Table 2.

[0318] Comparative Example 16

[0319] In this example, kiwifruit root tissue cell nuclei were prepared using KR3 lysate components including 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m / v) digitonin (Sigma, 11024-24-1). The remaining operating steps were the same as those in Comparative Example 14.

[0320] The results are shown in Table 2.

[0321] Comparative Example 17

[0322] In this example, kiwifruit root tissue cell nuclei were prepared using KR4 lysate consisting of 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (v / v) Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, Sigma, 9036-19-5). The remaining steps were the same as those in Comparative Example 14.

[0323] The results are shown in Table 2.

[0324] Comparative Example 18

[0325] In this example, kiwifruit root tissue nuclei were prepared using KR5 lysate consisting of 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2). The remaining steps were the same as those in Comparative Example 14.

[0326] The results are shown in Table 2.

[0327] Comparative Example 19

[0328] In this example, kiwifruit root tissue nuclei were prepared using KR6 lysate consisting of 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.1% (m / v) saponin (Nanjing Reagent, 8047-15-2). The remaining steps were the same as those in Comparative Example 14.

[0329] The results are shown in Table 2.

[0330] Comparative Example 20

[0331] In this example, kiwifruit root tissue nuclei were prepared using KR7 lysate consisting of 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2). The remaining steps were the same as those in Comparative Example 14.

[0332] The results are shown in Table 2.

[0333] Comparative Example 21

[0334] This example uses a double-sided blade to chop kiwifruit root tissue and KR5 lysis buffer for lysis.

[0335] 1) Prepare KR5 lysis buffer. Prepare the lysis buffer according to the following ingredients:

[0336] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysate on ice.

[0337] 2) Crushing samples.

[0338] Place 50 mg of kiwifruit root tissue sample in a petri dish on ice, cut the root into 0.5 mm segments using a double-sided blade, and transfer to a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis buffer pre-cooled on ice in step (1).

[0339] 3) Lyse cells.

[0340] Place the centrifuge tube in ice and incubate for 8 minutes.

[0341] 4) Microscopic examination and counting.

[0342] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0343] The results are shown in Table 3.

[0344] Comparative Example 22

[0345] This example uses scissors to cut the kiwifruit root tissue into pieces and then uses KR5 lysis solution for lysis. The operation is the same as that of Comparative Example 17.

[0346] The results are shown in Table 3.

[0347] Comparative Example 23

[0348] In this example, kiwifruit root tissue was homogenized and broken using a glass homogenizer, and lysed using KR5 lysis buffer.

[0349] 1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0350] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysate on ice.

[0351] 2) Crushing samples.

[0352] 50 mg of kiwifruit root tissue sample was transferred into a 1 mL glass homogenizer (Solarbio, YA0850), 1 mL of lysis solution pre-cooled on ice in step (1) was added, and the tissue was broken up by pounding 20 times with the glass homogenizer. The liquid in the homogenizer was transferred into a 2 mL centrifuge tube (Axygen, MCT-200-C).

[0353] 3) Lyse cells.

[0354] Place the centrifuge tube in ice and incubate for 8 minutes.

[0355] 4) Microscopic examination and counting.

[0356] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0357] The results are shown in Table 3.

[0358] Comparative Example 24

[0359] In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR5 lysis buffer.

[0360] 1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0361] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysate on ice.

[0362] 2) Crushing samples.

[0363] Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Rapid tissue low temperature crushing homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Add steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0364] 3) Lyse cells.

[0365] Place the centrifuge tube in ice and incubate for 8 minutes.

[0366] 4) Microscopic examination and counting.

[0367] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0368] The results are shown in Table 3.

[0369] Comparative Example 25

[0370] This example uses liquid nitrogen to grind and crush kiwifruit root tissue, and KR5 lysis buffer for lysis.

[0371] 1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0372] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysate on ice.

[0373] 2) Crushing samples.

[0374] 50 mg of kiwi root tissue sample was transferred into a mortar and a certain amount of liquid nitrogen was added to grind until the tissue was completely powdered. During this period, liquid nitrogen was added to keep the temperature low. The ground powder was transferred to a 2 mL centrifuge tube (Axygen, MCT-200-C) and 1 mL of the lysis solution pre-cooled on ice in step (1) was added.

[0375] 3) Lyse cells.

[0376] Place the centrifuge tube in ice and incubate for 8 minutes.

[0377] 4) Microscopic examination and counting.

[0378] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0379] The results are shown in Table 3.

[0380] Comparative Example 26

[0381] In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR8 lysis buffer.

[0382] 1) Prepare KR8 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0383] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.1U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0384] 2) Crushing samples.

[0385] Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Onebio high-throughput tissue grinder (onebio-48p). Add steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0386] 3) Lyse cells.

[0387] Place the centrifuge tube in ice and incubate for 8 minutes.

[0388] 4) Microscopic examination and counting.

[0389] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0390] 5) RNA quality inspection.

[0391] The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). The RNA was quality checked using an Agilent 2100 bioanalyzer.

[0392] The results are shown in Table 4

[0393] Comparative Example 27

[0394] In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR9 lysis buffer.

[0395] 1) Prepare KR9 lysis buffer. Prepare lysis buffer according to the following ingredients:

[0396] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.4U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0397] 2) Crushing samples.

[0398] Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Rapid tissue low temperature crushing homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Add steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0399] 3) Lyse cells.

[0400] Place the centrifuge tube in ice and incubate for 8 minutes.

[0401] 4) Microscopic examination and counting.

[0402] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the lysate in step (3), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0403] 5) RNA quality inspection.

[0404] The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). The RNA was quality checked using an Agilent 2100 bioanalyzer.

[0405] The results are shown in Table 4

[0406] Comparative Example 28

[0407] In this example, the kiwifruit root tissue was crushed using a crusher, and the KR10 lysis solution contained 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), and 0.6 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613). The remaining operations were the same as those in Comparative Example 25.

[0408] The results are shown in Table 5

[0409] Comparative Example 29

[0410] This example uses a 70 μm (FALCON, 352350) mesh to filter the cell nucleus suspension twice.

[0411] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0412] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0413] 2) Crushing samples.

[0414] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0415] 3) Lyse cells.

[0416] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0417] 4) Filter.

[0418] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0419] The lysate was aspirated one by one using a pipette and filtered through a 70 μm filter (FALCON, 352350). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0420] Note that all operations should be performed on ice.

[0421] 5) Filter.

[0422] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 70 μm filter (FALCON, 352350). Collect the filtrate into a new 15 mL centrifuge tube.

[0423] Note that all operations should be performed on ice.

[0424] 6) Microscopic examination and counting.

[0425] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the filtrate in step (5), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0426] The results are shown in Table 5

[0427] Comparative Example 30

[0428] In this example, the cell nucleus suspension was filtered once using a 30 μm (Miltenyi Biotec, 130-110-915) mesh, and the rest of the operations were the same as those in Comparative Example 29.

[0429] The results are shown in Table 5

[0430] Comparative Example 31

[0431] In this example, the cell nucleus suspension was filtered twice using a 30 μm (Miltenyi Biotec, 130-110-915) mesh, and the rest of the operations were the same as those in Comparative Example 29.

[0432] The results are shown in Table 5

[0433] Comparative Example 32

[0434] In this example, a 40 μm (FALCON, 352340) mesh was used to filter the cell nucleus suspension, and the rest of the operations were the same as those in Comparative Example 29.

[0435] The results are shown in Table 5

[0436] Comparative Example 33

[0437] In this example, the cell nucleus suspension was filtered twice using a 40 μm (FALCON, 352340) mesh, and the rest of the operations were the same as those in Comparative Example 29.

[0438] The results are shown in Table 5

[0439] Comparative Example 34

[0440] This example is based on filtering with a 70 μm (FALCON, 352350) mesh to remove calcium oxalate crystals.

[0441] 1) Prepare the lysis buffer. Prepare the KR lysis buffer according to the following ingredients:

[0442] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0443] 2) Crushing samples.

[0444] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0445] 3) Lyse cells.

[0446] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0447] 4) Filter.

[0448] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0449] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0450] Note that all operations should be performed on ice.

[0451] 5) Filter.

[0452] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0453] Note that all operations should be performed on ice.

[0454] 6) Filter.

[0455] The filtrate in step (5) was filtered through a 70 μm (FALCON, 352350) mesh, and the filtrate was collected into a new 15 mL centrifuge tube. The filtrate was placed on ice and filtered until the liquid was completely filtered into the 15 mL centrifuge tube.

[0456] 7) Microscopic examination and counting.

[0457] Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by DAPI staining under a microscope: Take 5 μL of the filtrate in step (6), mix it with DAPI staining in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.

[0458] The results are shown in Table 6

[0459] Comparative Example 35

[0460] In this example, a 40 μm (FALCON, 352340) sieve was used to filter and remove calcium oxalate crystals. The remaining operations were the same as those in Comparative Example 32.

[0461] The results are shown in Table 6

[0462] Comparative Example 36

[0463] In this example, calcium oxalate crystals were removed by filtration using a 30 μm (Miltenyi Biotec, 130-110-915) sieve, and the remaining operations were the same as those in Comparative Example 32.

[0464] The results are shown in Table 6

[0465] Comparative Example 37

[0466] This example is based on the removal of calcium oxalate crystals using 100×g differential centrifugation.

[0467] 1) Prepare the lysis buffer. Prepare the KR lysis buffer according to the following ingredients:

[0468] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0469] 2) Crushing samples.

[0470] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0471] 3) Lyse cells.

[0472] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0473] 4) Filter.

[0474] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0475] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0476] Note that all operations should be performed on ice.

[0477] 5) Filter.

[0478] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0479] Note that all operations should be performed on ice.

[0480] 6) Differential centrifugation.

[0481] The filtrate in step (5) was supplemented with pre-cooled PBS to 10 mL, and centrifuged at 4°C, 100×g, for 10 min. After centrifugation, the supernatant was transferred to a new centrifuge tube, and the precipitate was resuspended in 3 mL of pre-cooled PBS.

[0482] 8) Microscopic examination and counting.

[0483] The cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI stain: 5 μL of the supernatant and resuspension in step (6) were taken respectively, and mixed with DAPI stain in a ratio of 1:1. 10 μL was pipetted and added to the sample well of the hemocytometer for counting. The number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0484] The results are shown in Table 6

[0485] Comparative Example 38

[0486] This example is based on the use of density gradient centrifugation to remove calcium oxalate crystals.

[0487] 1) Prepare the lysis buffer. Prepare the KR lysis buffer according to the following ingredients:

[0488] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0489] 2) Crushing samples.

[0490] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0491] 3) Lyse cells.

[0492] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0493] 4) Filter.

[0494] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0495] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0496] Note that all operations should be performed on ice.

[0497] 5) Filter.

[0498] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0499] Note that all operations should be performed on ice.

[0500] 6) Density gradient centrifugation.

[0501] Prepare 50% (m / v) sucrose solution, 40% (m / v) sucrose solution, 20% (m / v) sucrose solution and 15% (m / v) sucrose solution respectively.

[0502] The filtrate obtained in step (5) was centrifuged at 4°C, 500×g, for 10 min. After centrifugation, the supernatant was discarded and the cell nucleus pellet was resuspended in 6 mL of 15% (m / v) sucrose solution.

[0503] In a Polyallomer centrifuge tube (Beckman, 355631), 50% (m / v) sucrose solution, 40% (m / v) sucrose solution, and 20% (m / v) sucrose solution were added from bottom to top, and then 6 mL of cell nucleus solution was slowly added to the top layer to form a density gradient.

[0504] The mixture was centrifuged at 18000 rpm for 90 min, and the 50% (m / v) sucrose solution layer, the 40% (m / v) sucrose solution layer and the 20% (m / v) sucrose solution layer were respectively placed in three new centrifuge tubes.

[0505] 7) Microscopic examination and counting.

[0506] The cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI stain: 5 μL of the 50% (m / v) sucrose solution layer, 40% (m / v) sucrose solution layer, and 20% (m / v) sucrose solution layer in step (6) were taken respectively, and mixed with the DAPI stain in a ratio of 1:1. 10 μL was pipetted and added to the sample well of the hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0507] The results are shown in Table 6

[0508] Comparative Example 39

[0509] This example is based on the removal of calcium oxalate crystals using flow cytometry.

[0510] 1) Prepare the lysis buffer. Prepare the KR lysis buffer according to the following ingredients:

[0511] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0512] 2) Crushing samples.

[0513] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0514] 3) Lyse cells.

[0515] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0516] 4) Filter.

[0517] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0518] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0519] Note that all operations should be performed on ice.

[0520] 5) Filter.

[0521] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0522] Note that all operations should be performed on ice.

[0523] 6) Flow cytometric sorting.

[0524] DAPI dye was added to the filtrate obtained in step (5), and the final concentration of DAPI was 5 μg / mL. The sorting condition was set to collect blue fluorescence signal (+) under ultraviolet excitation light.

[0525] 7) Microscopic examination and counting.

[0526] The cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI staining solution: 5 μL of the collected solution in step (6) was taken and mixed with DAPI staining solution in a ratio of 1:1. 10 μL was pipetted and added to the sample well of the hemocytometer for counting. The number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0527] The results are shown in Table 6

[0528] Comparative Example 40

[0529] This example is based on the use of an MS sorting column to remove calcium oxalate crystals.

[0530] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0531] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0532] 2) Crushing samples.

[0533] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0534] 3) Lyse cells.

[0535] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0536] 4) Filter.

[0537] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0538] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0539] Note that all operations should be performed on ice.

[0540] 5) Filter.

[0541] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0542] Note that all operations should be performed on ice.

[0543] 6) Collect the precipitate by centrifugation.

[0544] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0545] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0546] 7) Filter.

[0547] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0548] 8) Microscopic examination and counting.

[0549] The cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI staining solution: 5 μL of the collected solution in step (6) was taken and mixed with DAPI staining solution in a ratio of 1:1. 10 μL was pipetted and added to the sample well of the hemocytometer for counting. The number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0550] The results are shown in Table 6

[0551] Comparative Example 41

[0552] This example is based on the use of LS sorting columns to remove calcium oxalate crystals.

[0553] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0554] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0555] 2) Crushing samples.

[0556] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0557] 3) Lyse cells.

[0558] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0559] 4) Filter.

[0560] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0561] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0562] Note that all operations should be performed on ice.

[0563] 5) Filter.

[0564] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0565] Note that all operations should be performed on ice.

[0566] 6) Collect the precipitate by centrifugation.

[0567] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0568] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0569] 7) Filter.

[0570] Add all 3 mL of the resuspension in step (6) to the LS Column (Miltenyi, 130-042-401), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0571] 9) Microscopic examination and counting.

[0572] The cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI staining solution: 5 μL of the collected solution in step (6) was taken and mixed with DAPI staining solution in a ratio of 1:1. 10 μL was pipetted and added to the sample well of the hemocytometer for counting. The number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0573] The results are shown in Table 6

[0574] Comparative Example 42

[0575] The washing conditions in this embodiment are 100×g, 4° C., and 10 min per wash.

[0576] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0577] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0578] 2) Crushing samples.

[0579] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0580] 3) Lyse cells.

[0581] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0582] 4) Filter.

[0583] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0584] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0585] Note that all operations should be performed on ice.

[0586] 5) Filter.

[0587] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0588] Note that all operations should be performed on ice.

[0589] 6) Collect the precipitate by centrifugation.

[0590] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0591] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0592] 7) Filter.

[0593] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0594] 8) Collect the precipitate by centrifugation.

[0595] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 100×g for 10 minutes at 4°C.

[0596] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0597] 9) Quality inspection, microscopic examination and counting.

[0598] The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0599] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (8) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0600] The results are shown in Table 7

[0601] Comparative Example 43

[0602] The washing conditions in this embodiment are 1000×g, 4° C., and 10 min per wash.

[0603] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0604] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0605] 2) Crushing samples.

[0606] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0607] 3) Lyse cells.

[0608] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0609] 4) Filter.

[0610] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0611] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0612] Note that all operations should be performed on ice.

[0613] 5) Filter.

[0614] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0615] Note that all operations should be performed on ice.

[0616] 6) Collect the precipitate by centrifugation.

[0617] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0618] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0619] 7) Filter.

[0620] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0621] 8) Collect the precipitate by centrifugation.

[0622] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 1000×g for 10 minutes at 4°C.

[0623] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0624] 9) Quality inspection, microscopic examination and counting.

[0625] The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0626] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (8) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0627] The results are shown in Table 7.

[0628] Comparative Example 44

[0629] The washing conditions in this embodiment are 300×g, 4° C., and 10 min per wash.

[0630] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0631] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0632] 2) Crushing samples.

[0633] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0634] 3) Lyse cells.

[0635] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0636] 4) Filter.

[0637] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0638] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0639] Note that all operations should be performed on ice.

[0640] 5) Filter.

[0641] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0642] Note that all operations should be performed on ice.

[0643] 6) Collect the precipitate by centrifugation.

[0644] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0645] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0646] 7) Filter.

[0647] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0648] 8) Collect the precipitate by centrifugation.

[0649] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0650] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0651] 9) Quality inspection, microscopic examination and counting.

[0652] The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0653] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI stain: 5 μL of the resuspension in step (8) was mixed with DAPI stain in a ratio of 1:1, 10 μL was pipetted into the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.

[0654] The results are shown in Table 7

[0655] Comparative Example 45

[0656] The washing conditions in this example are 300×g, 4° C., and 10 min for three washes.

[0657] 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients:

[0658] 2% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2U / mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysate on ice.

[0659] 2) Crushing samples.

[0660] Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.

[0661] 3) Lyse cells.

[0662] After the instrument stops completely, take out the centrifuge tube and put it in ice, and incubate it for 7 minutes.

[0663] 4) Filter.

[0664] Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute.

[0665] The lysate was aspirated one by one using a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected in a new 50 mL centrifuge tube (Corning, 430828).

[0666] Note that all operations should be performed on ice.

[0667] 5) Filter.

[0668] Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.

[0669] Note that all operations should be performed on ice.

[0670] 6) Collect the precipitate by centrifugation.

[0671] Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0672] After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.

[0673] 7) Filter.

[0674] Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.

[0675] 8) Collect the precipitate by centrifugation.

[0676] Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0677] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0678] 9) Collect the precipitate by centrifugation.

[0679] Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0680] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.

[0681] 10) Collect the precipitate by centrifugation.

[0682] Place the centrifuge tube in step (9) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.

[0683] After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.

[0684] 11) Quality inspection, microscopic examination and counting.

[0685] The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-volume spectrophotometer and a fluorescence spectrophotometer.

[0686] The concentration of live cell nuclei, the ratio of agglomeration, and the ratio of impurities were calculated by microscopic examination using DAPI staining solution: 5 μL of the resuspension solution in step (10) was taken and mixed with DAPI staining solution in a ratio of 1:1. 10 μL of the resuspension solution was pipetted and added to the sample well of the hemocytometer for counting. The number of cell nuclei in the counting area was observed under a microscope. Fluorescence was shown as cell nuclei.

[0687] The results are shown in Table 7

[0688] Comparative Example 46

[0689] The experimental sample in this example is 50 mg of Arabidopsis leaves, and the specific operation is the same as that in Example 1.

[0690] The results are shown in Table 8

[0691] Comparative Example 47

[0692] The experimental sample in this example is 50 mg of Arabidopsis roots, and the specific operation is the same as that in Example 1.

[0693] The results are shown in Table 8

[0694] Comparative Example 48

[0695] The experimental sample in this example is 50 mg corn embryo, and the specific operation is the same as that in Example 1.

[0696] The results are shown in Table 8

[0697] Table 1 Results of Examples 1 to 4

[0698]

[0699] The present invention describes a method suitable for sequencing single cell nuclei of kiwifruit root tissue, including a method for extracting cell nuclei from kiwifruit root tissue and a method for optimizing cell nuclei suspension. The technical method provided by the present invention can introduce single cell sequencing technology into the relevant research on kiwifruit molecular mechanism and breeding, and can effectively and efficiently promote the progress of research.

[0700] The invention discloses a method for preparing single cell nuclei suitable for kiwifruit root tissue, which mainly comprises a lysate, the components of which are 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), and 0.2 to 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613). The cell nuclei are extracted from the tissue by crushing the sample and lysing the cells, and then the cell nuclei are obtained after suspension optimization treatment such as sieve filtration and centrifugal washing to obtain a cell nucleus greater than 1×10 6 The cell nucleus suspension with a fragmentation rate of less than 10% and a clumping rate of less than 4% meets the quality requirements of 10× single-cell sequencing for cell nucleus suspension. The purpose of using high-throughput single-cell sequencing technology to study kiwifruit root tissue was achieved.

[0701] In order to obtain a method suitable for single-cell nuclear sequencing of kiwifruit root tissue in the present invention, a series of explorations were conducted from the aspects of lysate formula, experimental operation steps, optimization methods, etc., and the content of the present invention was finally obtained by consulting materials and verification. First, by comparing Examples 1 to 3, the method in "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" was used to prepare the nuclear suspension of Arabidopsis leaves, kiwifruit leaves, and kiwifruit root tissues, respectively. The results showed that the method in the document can only complete the preparation of the nuclear suspension of Arabidopsis leaf tissue, and cannot complete the preparation of the nuclear suspension of kiwifruit leaves and kiwifruit root tissues; and the proportion of impurities in the prepared Arabidopsis leaf nuclear suspension is slightly higher. Although it meets the minimum requirements for single-cell sequencing for samples, there are risks in single-cell sequencing. In Comparative Examples 4 to 6, the method of "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" was used to extract nuclei using existing commercial kits to prepare nucleus suspensions of Arabidopsis root tissue, kiwi leaf tissue, and kiwi root tissue. The results showed that the method in the literature can only complete the preparation of nucleus suspensions of Arabidopsis root tissue, but cannot complete the preparation of nucleus suspensions of kiwi leaf tissue. The results showed that the number of kiwi root nuclei extracted by the existing commercial kits was very small, and it was impossible to successfully prepare a kiwi root tissue nucleus suspension that met the requirements of single cell sequencing. Both of the above methods showed the phenomenon of preparing a small number of nuclei and a large number of impurities. The key to ensuring the number of extracted nuclei is that the lysis solution fully lyses the tissue while having a suitable buffer to ensure the integrity of the nuclei.

[0702] First, by comparing Examples 7 to 20, the buffer system suitable for kiwifruit root tissue was explored. The results are shown in Table 1. The buffer system in the existing scheme is not suitable for the kiwifruit root tissue cell nucleus extraction experiment; kiwifruit grows in the wild, and the cell growth mechanism is very different from that of the model organism. Calcium oxalate, aldehydes, etc. are enriched in kiwifruit root cells. Compared with the buffer components that require stronger buffering capacity such as the model organism Arabidopsis, among the commonly used buffer components, Tris-HCl, MOPS, etc. are not applicable. Increasing the concentration will promote the reaction of Tris with aldehydes and various enzymes in the solution, resulting in an unstable lysis system. Finally, after exploration, a buffer system constructed with L-tartaric acid and sodium citrate was selected. L-tartaric acid is commonly used as an acidulant for beverages and other foods, has a strong buffering capacity, and plays a major buffering role in the present invention; at the same time, tartaric acid also has a strong reducing property, and plays a protective role for nucleic acids in the cell nucleus in the present invention. The results of the comparative example show that when the concentration of the buffer component is low, the pH of the buffer cannot be kept stable, and the nucleus is broken due to the change of the pH of the buffer; when the concentration of the buffer component is high, the nucleus agglomerates due to the high ion concentration in the buffer, resulting in experimental failure. Therefore, the buffer component for preparing the nucleus in the present invention is 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15 to 25 mM sodium citrate.

[0703] Extracting plant cell nuclei requires destroying the cell membrane, and the basic structure of the cell membrane is a phospholipid bilayer. It is common to use detergents to destroy the phospholipid bilayer to achieve the purpose of releasing the cell nucleus. In comparative examples 7 to 10, the lysate formula was explored, and experiments were conducted using lysate components with different properties and working principles. The results showed that when strong detergents SDS (sodium dodecyl sulfate) and sodium deoxycholate were used as lysate components, a large number of cell nuclei were broken, and it was impossible to collect a sufficient number of intact cell nuclei; when weak detergent digitonin was used as a lysate component, since digitonin could not dissolve membrane proteins, the cell membrane structure could not be fully destroyed, and relatively more cell nuclei could be collected, but the requirement for the number of cell nuclei for single-cell sequencing could not be met. Triton X-100 is a mild detergent derived from polyethylene oxide and contains an alkylphenyl hydrophobic group. Its ability to bind lipid molecules is between sodium dodecyl sulfate and digitonin, and it can be used as a cosolvent for membrane proteins. The number of nuclei obtained by lysis is more than that of digitonin, but Triton X-100 will damage the nuclear membrane while acting on the cell membrane, and it is still impossible to obtain enough nuclei for single cell nuclear sequencing experiments. The commonly used method of preparing nuclei by dissolving cell membranes with detergents is not suitable for the preparation of nuclei of kiwi root tissue. Saponin is commonly used in pharmaceutical raw materials, mainly for the synthesis of hormone drugs. Saponin is composed of saponin and sugar, uronic acid or other organic acids, and saponin gives saponin the function of emulsifying lipid molecules. The results of the comparative example show that when saponin is used as a lysis component, the yield of nuclei is significantly improved compared with SDS, Triton X-100, etc.; at the same time, because saponin does not cause protein denaturation, saponin as a lysis component will not damage the nuclear membrane of the cell nucleus. The results of the comparative example show that when the saponin concentration is lower than 2%, the number of cell nuclei will decrease significantly, because saponin itself contains sugars and organic acids, etc. When the saponin concentration is higher than 4%, it will affect the stability of the lysis system and reduce the number of cell nuclei. Therefore, the concentration range of saponin in the lysis solution of the present invention is 2% to 4% (m / v).

[0704] Table 2 Comparative Examples 1 to 20

[0705]

[0706]

[0707]

[0708]

[0709]

[0710] Plant cell membranes are wrapped by cell walls, which are composed of cellulose, hemicellulose, pectin and other components. Kiwi plants are large deciduous vines, and the supporting strength of the cell walls is much higher than that of Arabidopsis. Therefore, in the method of breaking the cell walls, stronger breaking conditions are needed to help obtain more nuclei. Next, by comparing the effects of different treatment methods including blade chopping, scissors chopping, homogenization, crusher crushing, mortar grinding, etc. on the release of nuclei, the results show that the number of nuclei obtained by the experimental methods of blade chopping, scissors chopping, and mortar grinding is lower than that of the homogenization method. Mortar liquid nitrogen grinding is the most sufficient way to break tissue, but mortar grinding also damages the nucleus while breaking the cell wall. The results of the comparative examples show that the crusher crushing method assists the lysate to fully release the nucleus while causing less damage to the nucleus, which is the best method for extracting the nucleus of kiwi root tissue.

[0711] Table 3 Comparative Examples 21 to 25

[0712]

[0713] When single-cell transcriptome experiments are performed on plant cell nuclei, the integrity of intracellular mRNA needs to be ensured. After the cells are broken, the RNase in the cytoplasm is released into the buffer system to degrade the mRNA. Therefore, RNase inhibitors and reducing agents are needed to protect nuclear mRNA. The RNase content in different species and tissues varies greatly. The results of comparative examples 26 to 28 show that for 50 to 100 mg of kiwi root tissue, 0.2 to 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613) can well protect the integrity of mRNA. There is no significant difference in the results obtained by increasing the RNase inhibitor, which will also increase costs and waste reagents. Therefore, the concentration of RNase inhibitor in KR lysate is 0.2 to 0.4 U / mL.

[0714] Table 4. Comparative Examples 26 to 28

[0715]

[0716] The crude cell nucleus suspension obtained by extracting the tissue after being treated with a lysate contains a large amount of impurities such as cell fragments and mRNA and organelles released by cell fragmentation. Under such conditions, the cell nucleus suspension has large fragments and cannot be used for single-cell sequencing experiments. The large fragments need to be removed to meet the requirements of single-cell sequencing. First, the larger tissue and cell fragments are filtered to remove them. By comparing Examples 29 to 33, the yield and impurity removal efficiency of the crude cell nucleus suspension filtered with different aperture meshes and different filtering times are verified. The results of Comparative Example 29 show that the 70μm mesh cannot achieve the purpose of impurity removal very well. The impurities of the suspension after filtration with the 70μm mesh are not significantly reduced, and there is still no obvious effect after two filtrations. The sample broken by the crusher has fewer fragments larger than 70 μm. In comparative examples 30-31, the effect of filtering the crude cell nucleus suspension of kiwifruit root with a 30 μm aperture sieve was verified. The results showed that the 30 μm cell sieve would lead to a large loss of cell nuclei, and the filtration process was affected by the high fragmentation rate, and the filtration time was long, which increased the risk of RNA degradation in the cell nucleus. Therefore, the 30 μm sieve could not be used as a method for purifying the crude cell nucleus suspension of kiwifruit root. The results of comparative examples 32-33 show that the 40 μm cell sieve can quickly remove impurities in the crude cell nucleus suspension. The best method for purifying the crude cell nucleus suspension of kiwifruit root is to filter twice with a 40 μm cell sieve, with an impurity removal efficiency of up to 80%, and a cell nucleus yield of more than 70%. This choice not only quickly and effectively reduces the fragmentation rate, but also has a higher cell nucleus yield.

[0717] Table 5 Comparative Examples 29 to 33

[0718]

[0719] During the growth of kiwifruit plants, a large number of calcium oxalate needle crystals will accumulate in cells / tissues. These calcium oxalate needle crystals are an important component of plant defense and self-protection. These calcium oxalate crystals will be released into the cell nucleus suspension after the tissue is broken ( Figure 3The figure shows needle-shaped calcium oxalate crystals in the cell nucleus suspension). There are still needle-shaped crystals in the cell suspension after screen filtration. In the screen filtration, neither 30μm nor 70μm can effectively complete the filtration of this type of impurities. The presence of calcium oxalate crystals will make it impossible to carry out single-cell sequencing. Calcium oxalate crystals exist in kiwi root tissue as needle-shaped crystals, with different thicknesses and lengths. According to microscopic observation, the length of calcium oxalate crystals is greater than 70μm. The effect of 70μm cell sieve on removing calcium oxalate crystals was verified by comparative example 34, and the results showed that 70μm cell sieve could not effectively remove needle-shaped calcium oxalate crystals. The effect of 40μm and 30μm sieves on removing calcium oxalate crystals was verified by comparative examples 35 to 36, and the results showed that reducing the sieve aperture still could not remove calcium oxalate crystals. Needle-shaped calcium oxalate crystals can easily pass through the sieve with liquid flow, so the method of sieve filtration cannot remove needle-shaped calcium oxalate crystals. Next, by comparing Examples 37 to 39, the effects of differential centrifugation, density gradient centrifugation, and flow cytometry on removing calcium oxalate crystals were verified, and the results showed that differential centrifugation could not remove calcium oxalate crystals and would cause a large loss of nuclei; density gradient centrifugation would cause a large loss of nuclei, and there would be risks in continuing single-cell nuclear sequencing, as the density of impurities was similar to that of nuclei, resulting in the inability of density gradient centrifugation to purify nuclei well, and both differential centrifugation and density gradient centrifugation could not achieve the purpose of purifying nuclei. The presence of needle-like calcium oxalate crystals would directly cause the flow cytometer to be blocked, and it would be impossible to collect enough nuclei for the experiment. Comparative Examples 40 to 41 use MACS sorting columns to explore the optimization of cell nucleus suspensions. Sorting columns (MACS) are usually used in magnetic fields to bind specific antigens on the surface of target cell membranes through antibodies with magnetic beads, so as to be used for sorting specific cell types of animals. In the present invention, in a non-magnetic field, the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized. Small cell nuclei can pass through the sorting column with the liquid flow, while needle-shaped calcium oxalate crystals will be left in the sorting column and cannot pass through with the liquid flow. The purpose of purifying cell nuclei can achieve the purpose of removing impurities while ensuring the cell nucleus yield, solving the problem of optimizing single cell nucleus suspensions. The results show that all indicators of the filtrate obtained by filtering the single cell nucleus suspension through the MS sorting column meet the requirements of the 10×Genmics platform for single-cell sequencing.

[0720] Table 6. Comparative Examples 34 to 41

[0721]

[0722]

[0723] After the calcium oxalate crystals are removed, the free mRNA in the cell nucleus suspension needs to be removed by washing to improve the data quality. At the same time, the concentration of the cell nucleus suspension needs to be adjusted. In Comparative Examples 42 to 45, the washing conditions were verified. The results show that too small a centrifugal force will lead to a large loss of cell nuclei, too large a centrifugal force will lead to cell nucleus damage, and too many washing times will lead to cell nucleus fragmentation; the washing conditions are 4°C, 300×g to 500×g centrifugation for 10 minutes, washing twice, and the free mRNA in the cell nucleus suspension is basically removed. At the same time, the cell nucleus yield is higher than other methods, which is suitable for the washing conditions of kiwi root tissue cell nucleus suspension.

[0724] Table 7. Comparative Examples 42 to 45

[0725]

[0726]

[0727] Plant cell walls of different species and tissues have different compositions, and the composition of cell contents is also different. Comparative Examples 46 to 48 verified the results of preparing the nuclear suspension of Arabidopsis leaves, Arabidopsis root tips, and corn embryos using the KR lysate and the cell nucleus extraction and optimization methods in this patent. The results show that the cell nucleus preparation method in this patent, as well as the corresponding lysate formula and cell nucleus optimization method, correspond one-to-one with kiwifruit root tissues, and the steps need to be performed in sequence to complete the preparation of the nuclear suspension of kiwifruit root tissues, which is not applicable to other species and tissues.

[0728] Table 8. Comparative Examples 46 to 48

[0729] In summary, the present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing. By comparing Examples 1 to 28, a KR lysate solution is obtained, which comprises 2% to 3% (v / v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m / v) saponin (Nanjing Reagent, 8047-15-2), 0.2 to 0.4 U / mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATEBIOLO; the method of assisting the release of kiwifruit root nuclei is crushing with a crusher. By comparing Examples 29 to 41, an experimental method for optimizing the nucleus suspension was obtained: filtering twice with a 40μm sieve to remove large fragments, and removing calcium oxalate needle crystals with an MS sorting column; by comparing Examples 42 to 45, a method for washing a kiwifruit root tissue nucleus suspension with a high nucleus yield and removal of environmental RNA was verified, which was washed twice at 300 to 500×g, 4°C, and 10min. The results of Comparative Examples 46 to 48 show that the KR lysate, nucleus suspension filtration operation, and nucleus suspension washing operation described in the present invention are only applicable to the preparation of kiwifruit root tissue nucleus suspension.

[0730] The present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, which can effectively prepare the nucleus suspension required for the single-cell transcriptome sequencing experiment of the 10x Genomics platform. In the method for preparing a kiwifruit nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention, there is a one-to-one correspondence between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results, that is, there is a compatibility relationship between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results. For example, the kiwifruit root, KR lysate, and nucleus suspension filtration operation mentioned in Examples 1 to 4 of the present invention should be used according to the method specified in the present invention to effectively prepare the nucleus suspension, and the various indicators of the prepared nucleus suspension can meet the requirements of single-cell transcriptome sequencing. The method for preparing a kiwifruit root cell nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention is scientifically rigorous in operation, highly repeatable, and can effectively and efficiently separate a cell nucleus suspension with a sufficient number, low impurity rate, and extremely low environmental RNA from kiwifruit root tissue, and the obtained cell nucleus suspension can meet various indicators of single-cell transcriptome sequencing on the 10x Genomics platform.

[0731] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, characterized in that: The specific steps include: 1) Prepare KR lysis buffer: First, prepare KR lysis buffer and precool the prepared solution; the KR lysis buffer contains the following ingredients: 2%~3% (v / v) L-tartaric acid, 15~25mM sodium citrate, 0.2~0.4% (m / v) saponin, and 0.2~0.4U / mL recombinant RNase inhibitor; 2) Sample crushing: transfer freshly collected or frozen kiwifruit root tissue samples into a centrifuge tube, add the pre-cooled KR lysis solution and steel beads, and crush the tissue on a crusher according to the instrument operating instructions; 3) Lyse cells: After disruption, place the centrifuge tube on ice and incubate for lysis; 4) Screen filtration: transfer the tissue lysate after lysis into a centrifuge tube, add pre-cooled PBS, let stand on ice for 1 minute, aspirate the tissue lysate, add it into the filter screen to filter and obtain the filtrate; 5) Screen filtration: Use the screen to filter the filtrate in step 4) again; 6) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 5), discard the supernatant, and resuspend the precipitate in PBS; 7) Filtration: Add all the precipitate resuspension in step 6) into the separation column for filtration; 8) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 7), discard the supernatant, and resuspend the precipitate in PBS.

2. The preparation method according to claim 1, characterized in that In step (2), the disruptor is a Wanbo Biotech high-throughput tissue disruptor; the disrupting conditions are 1200-1300 rpm, 170-180 s.

3. The preparation method according to claim 1, characterized in that: In step (3), the incubation time is 7 to 8 minutes.

4. The preparation method according to claim 1, characterized in that: In step (4), the filter is a FALCON 40 μm Cell Strainer with a pore size of 40 μm; and the filtration is performed at 0-5°C throughout the entire process.

5. The preparation method according to claim 1, characterized in that: In step (6), the centrifugation temperature is 4-6°C, the centrifugation time is 10-15 minutes, and the centrifugal force is 300-500×g.

6. The preparation method according to claim 1, characterized in that: In step (7), the separation column is a Miltenyi MS Column; and the filtration is performed at 0-5°C throughout the entire process.

7. The preparation method according to claim 1, characterized in that: In step (8), the centrifugation temperature is 4-6°C, the centrifugation time is 10-15 minutes, and the centrifugal force is 300-500×g.

8. A KR lysis solution or a reagent / kit containing the KR lysis solution for use in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, kiwifruit root tissue nuclei transcriptome sequencing, and kiwifruit root tissue nuclei ATAC sequencing research; the KR lysis solution comprises the following ingredients: 2%~3% (v / v) L-tartaric acid, 15~25mM sodium citrate, 0.2~0.4% (m / v) saponin, and 0.2~0.4U / mL recombinant RNase inhibitor.

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