A kit and method for extracting nuclei from plant seed grains

Abstract

The application discloses a kit and a method for extracting nuclei in plant fruit kernels. The kit comprises a lysis solution, a washing solution and a purification solution. The lysis solution comprises 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / muL RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS and 200-350 mM sucrose. The washing solution comprises 2% w / v BSA, 1.25-2.5 mM DTT and 0.2-0.4 U / muL RNase inhibitor. The purification solution comprises methanol and at least two Percoll solutions with different concentrations. The kit with specific components and the optimized extraction method can quickly obtain a high-purity nucleus suspension without extracting protoplasts, effectively remove impurities and secondary metabolites, reduce the risk of RNA degradation, and are simple to operate and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of cell nucleus extraction technology, specifically relating to a kit and method for extracting cell nuclei from plant fruit seeds. Background Technology

[0002] With the development of single-cell technology, it has become an important tool for mapping and analyzing plants. Plant cell nuclei are typically extracted using two conventional methods: one is to extract the protoplast first and then the nucleus, and the other is to extract the nucleus directly. However, the protoplast dissociation method requires numerous materials, is complex and costly, has a long experimental time, and carries a greater risk of nucleus degradation. Conventional direct extraction methods often fail to produce clean nucleus suspensions, easily introducing a large number of impurities during the extraction process, resulting in nucleus suspensions that do not meet the requirements of downstream experiments. This is especially true for seed samples, whose unique structure often results in a high level of impurities when extracting nuclei using existing techniques, making them unsuitable for single-cell sequencing. Therefore, there is an urgent need for a method that can obtain relatively clean nuclei without extracting protoplasts, addressing the problems of complex operation, long processing time, and high impurity levels in existing techniques. Summary of the Invention

[0003] To address at least one of the aforementioned problems, this invention provides a kit and method for extracting cell nuclei from seeds in plant fruits; by employing a specific combination of lysis buffer, washing buffer, and purification buffer, along with optimized extraction steps, high-purity cell nuclei can be extracted rapidly and efficiently.

[0004] To achieve the above objectives, the present invention employs the following technical means: A kit for extracting cell nuclei from plant fruit seeds, the kit comprising a lysis buffer, a washing buffer, and a purification buffer; the lysis buffer comprising 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / μL RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS, and 200-350 mM sucrose; the washing buffer comprising 2% w / v BSA, 1.25-2.5 mM DTT, and 0.2-0.4 U / μL RNase inhibitor; and the purification buffer comprising methanol and at least two different concentrations of Percoll solution.

[0005] The above-mentioned method, through the synergistic effect of specific components of the lysis buffer, washing buffer and purification buffer, can directly lyse seed cells and effectively remove impurities without extracting protoplasts, obtaining a high-purity cell nuclear suspension, which significantly shortens the experimental time and reduces the risk of RNA degradation in the cell nucleus.

[0006] As one implementation method, the methanol is pre-cooled to -20°C before use.

[0007] The above method, through pre-cooled methanol treatment, can effectively remove secondary metabolites and fix the cell nucleus, making it less prone to degradation and further improving the quality of extracted cell nuclei.

[0008] The step of processing with methanol is performed before separation with Percoll solution.

[0009] In one embodiment, the Percoll solution comprises a Percoll solution with a concentration of 60-65% v / v and a Percoll solution with a concentration of 25-30% v / v.

[0010] In one embodiment, the Percoll solution is diluted with the cleaning solution.

[0011] In addition, the present invention also provides a method for extracting cell nuclei from plant fruit seeds, comprising the following steps: S1. Take the seeds from the plant fruit, wash them with PBS and then dry them. S2. Add the lysis solution to the clean grains until they are completely submerged, and mechanically cut them into a homogenous slurry of uniform size using a pre-cooled blade. S3. Add the cleaning solution to the obtained homogenate to terminate the pyrolysis, and blow the homogenate with a Pasteur pipette. S4. Collect the obtained homogenate through a cell sieve into a new centrifuge tube, centrifuge, discard the supernatant, add the washing solution to resuspend the precipitate, centrifuge again, and aspirate the supernatant to obtain the first crude suspension. S5. Take the residue from the cell sieve, transfer it to a new sample tube, add the lysis buffer, cut it with a pre-cooled blade for 5-10 min, blow it with a Pasteur pipette for 2-3 min, then pass it through the cell sieve to collect the suspension. Centrifuge and discard the supernatant. Add washing buffer to resuspend the precipitate, centrifuge, and aspirate the supernatant to obtain the second coarse suspension. S6. Combine the first coarse suspension and the second coarse suspension to obtain a mixed suspension; S7. After centrifuging the mixed suspension, discard the supernatant, add methanol pre-cooled at -20 ℃ to resuspend the precipitate, and then fix it at -20 ℃; centrifuge to enrich the precipitate, resuspend it with the washing solution, centrifuge again, wash away the methanol component, and add the washing solution to the precipitate to resuspend the precipitate. S8. Add Percoll solution of different concentrations to the sample tube in descending order of concentration, then add the resuspended cell nucleus suspension to form a discontinuous liquid layer, and centrifuge. S9. Aspirate the liquid from the second layer and transfer it to a sample tube containing the washing solution. After centrifugation and enrichment, resuspend the precipitate using the washing solution to obtain a pure cell nucleus suspension.

[0012] The above scheme releases cell nuclei by combining mechanical shearing and chemical lysis, and improves the yield of cell nuclei by secondary extraction of filter residue; purification is carried out by pre-cooled methanol and Percoll density gradient centrifugation, which effectively removes impurities and secondary metabolites, and obtains a high-quality cell nucleus suspension that meets the requirements of single-cell sequencing.

[0013] In one implementation, in step S8, the volumes of the three liquids—Percoll solution of different concentrations and crude cell nucleus suspension—are all the same.

[0014] The above scheme, by controlling the volume ratio of each liquid layer, helps to form a stable density gradient interface, thereby more accurately separating cell nuclei from impurities and improving separation efficiency.

[0015] In one implementation, the centrifugation parameters in steps S4 and S5 are the same: 4℃, 500-600 rcf, centrifugation for 5-10 min; 4℃, 50-100 rcf, centrifugation for 1 min.

[0016] The above method, through specific centrifugation parameter settings, can effectively collect cell nuclei while avoiding excessive centrifugal force that could damage cell nuclei or cause impurity precipitation, thus ensuring the integrity and purity of cell nuclei.

[0017] In one implementation, the centrifugation parameters in steps S7 and S9 are 4°C, 500-600 rcf, and centrifugation for 5 min.

[0018] The above scheme simplifies the operation process by using standardized centrifugation conditions, while ensuring effective enrichment and washing of cell nuclei.

[0019] In one implementation, in step S8, the centrifugation parameters are 4°C, 1000-2000 rcf, 5-10 min centrifugation, and the centrifuge acceleration and deceleration rates are 4 for acceleration and 4 for deceleration.

[0020] The above method uses high centrifugal force to perform density gradient centrifugation, ensuring that cell nuclei can effectively pass through the density medium layer and accumulate at specific locations, thus achieving complete separation from light impurities.

[0021] In addition, the present invention also provides a suspension of cell nuclei from plant fruit seeds, which is prepared by any of the methods described above.

[0022] The above solution provides a high-purity, high-integrity cell nuclear suspension product that can be directly applied to downstream experiments such as single-cell sequencing, and has broad application prospects.

[0023] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: This invention provides a kit and method for rapidly extracting clean cell nuclei from seeds, offering advantages such as convenience, speed, and low cost. By using a chemical lysis buffer to assist mechanical lysis, the cell nuclei are released from the hardened seeds. A secondary extraction of the filter residue significantly improves the yield of cell nuclei. In particular, a two-step purification process using pre-cooled methanol and Percoll density gradient centrifugation effectively removes impurities such as secondary metabolites, resulting in a relatively clean cell nucleus suspension. This method obtains relatively clean cell nuclei without extracting protoplasts, significantly shortening experimental time and reducing the risk of RNA degradation within the cell nucleus due to complex and time-consuming experiments. Compared to conventional flow cytometry methods for sorting cell nuclei, this method requires less equipment, reduces experimental expenses, and avoids cell nucleus loss caused by low flow cytometry yields. It is particularly suitable for plants containing components that may affect flow cytometry fluorescence. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the Percoll density gradient centrifugation stratification before and after in Embodiment 3 of the present invention; Figure 2 This is a schematic diagram of the results of trypan blue microscopy on a cell nucleus suspension in Example 3 of the present invention; Figure 3 This is a schematic diagram comparing the morphology of cell nuclei under different lysis buffer concentrations in Example 5 of the present invention. The left figure shows the microscopic examination results of cell nuclei in the 0.5% w / v Triton X100 group, and the right figure shows the microscopic examination results of the 1% w / v Triton X100 group.

[0025] Figure 4 It is the crude suspension mixture treated with methanol in Example 4 of this invention; Figure 5 This is a diagram showing the effect of gradient separation performed directly without methanol treatment in Example 6 of the present invention. Figure 6 The images show the separation effects of different Percoll solution combinations in Example 7 of this invention. The left image shows the effect of centrifugation separation of the first group, and the right image shows the effect of centrifugation separation of the second group. Figure 7 This is the separation effect under different Percoll solution combinations in Example 7 of the present invention. The left figure shows the microscopic examination results of the second group, and the right figure shows the microscopic examination results of the third group. Figure 8This is a diagram showing the separation effect of the centrifuge under the conditions of raising and lowering the centrifuge by 9 degrees in Embodiment 8 of the present invention. Detailed Implementation

[0026] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0028] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0029] Example 1 This embodiment provides a kit for extracting cell nuclei from plant fruit seeds. The kit includes a lysis buffer, a washing buffer, and a purification buffer.

[0030] The lysis buffer is the core component of the kit, and its formulation includes 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / μL mouse RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS, and 200-350 mM sucrose.

[0031] In this embodiment, the murine RNase inhibitor was produced by Yisheng Biotechnology (Shanghai) Co., Ltd., MurineRNase Inhibitor, 40 U / μL, catalog number: 10603ES60.

[0032] In this lysis buffer system, Triton X100, as a nonionic surfactant, plays a crucial role in lysing the cell membrane and releasing the cell nucleus. Through extensive experiments, the inventors discovered that controlling the concentration of Triton X100 is critical: if the concentration is below 0.04% w / v, the tough cell walls and membranes of plant seeds result in weak lysis, failing to effectively release the cell nucleus and leading to extremely low yields; while if the concentration is above 0.4% w / v, although lysis efficiency increases, excessive surface activity can damage the nuclear membrane structure, causing nucleus rupture and leakage of contents, severely impacting the quality of subsequent single-cell sequencing. Therefore, limiting Triton X100 to the range of 0.04%-0.4% w / v ensures sufficient release of the cell nucleus while maximizing the preservation of its morphological integrity. Furthermore, RNase inhibitors and dithiothreitol (DTT) are specifically added to the lysis buffer. RNase inhibitors effectively suppress the activity of endogenous RNases, preventing RNA degradation in the cell nucleus and ensuring the accuracy of downstream molecular experiments. DTT acts as a reducing agent, maintaining a reducing environment in the solution and further protecting the stability of nucleic acids and proteins. The addition of spermine and spermidine helps maintain the compact structure of chromatin, preventing excessive chromatin disintegration during lysis and subsequent nuclear collapse. Sucrose and NaCl are used to regulate osmotic pressure, preventing the cell nucleus from swelling and bursting in a hypotonic environment or shrinking in an excessively high osmotic pressure. PBS serves as a buffer system, providing a stable pH environment.

[0033] The washing solution contains 2% w / v BSA, 1.25-2.5 mM DTT, and 0.2-0.4 U / μL of a mouse RNase inhibitor.

[0034] During the washing process, bovine serum albumin (BSA) plays a crucial sealing role, blocking non-specific binding sites on the contact surfaces of centrifuge tubes and pipette tips, thus reducing the physical adsorption loss of cell nuclei during the operation. Simultaneously, maintaining a certain concentration of DTT and RNase inhibitors in the washing solution ensures that while washing away residual impurities from the lysis buffer, it continuously provides a reducing environment and RNA protection environment for the cell nuclei, avoiding the risk of RNA degradation introduced during multi-step centrifugation.

[0035] The purification solution consists of methanol and 60-65% v / v Percoll solution and 25-30% v / v Percoll solution.

[0036] The Percoll solution is diluted with a washing solution, and the crude extract suspension is treated with methanol before separation using the Percoll solution.

[0037] Percoll is a polyvinylpyrrolidone-treated silica gel particle with low viscosity and low osmotic pressure, making it ideal for density gradient centrifugation of cells and organelles. By preparing Percoll solutions of different concentrations, discontinuous density gradient systems can be constructed, utilizing the density differences between cell nuclei and impurities such as cell debris and starch granules to achieve efficient separation. The introduction of methanol serves a dual function: firstly, as an organic solvent, methanol effectively removes hydrophobic secondary metabolites such as lipids and pigments abundant in plant seeds, which, if not removed, often interfere with subsequent microscopic observation and sequencing reactions; secondly, methanol can immobilize the cell nucleus, rapidly stabilizing the nuclear membrane and intranuclear structure, preventing nuclear degradation during prolonged centrifugation.

[0038] Through the specific formulation combination of the above-mentioned lysis buffer, washing buffer and purification buffer, the kit of this embodiment achieves synergistic effect among the components, and solves the technical problems of low yield, many impurities and easy degradation in the extraction of plant seed cell nuclei. The three reagents work together to efficiently release and purify highly intact cell nuclei from hard plant seed tissues.

[0039] Example 2 This embodiment further optimizes the composition of the purification solution in the kit, based on Example 1. Specifically, the methanol in the purification solution needs to be pre-cooled at -20°C before use.

[0040] In the process of extracting cell nuclei from plant fruit seeds, the applicant discovered that methanol pre-cooled to -20°C significantly improved the purity and quality of the final cell nucleus suspension compared to methanol stored at room temperature or 4°C. The technical mechanism is mainly reflected in the following two aspects: First, plant seeds are often rich in hydrophobic secondary metabolites such as lipids and pigments. If these substances remain in the cell nuclear suspension, they not only interfere with morphological observation under a microscope but may also inhibit enzyme activity in subsequent single-cell sequencing reactions, severely affecting experimental results. Methanol, as an organic solvent, has excellent lipid-dissolving ability. When samples are treated with methanol pre-cooled to -20°C, the low temperature environment increases the solubility of lipids in the organic solvent, more effectively extracting and removing impurities such as lipids and pigments from the cell nuclear suspension, thereby significantly reducing background noise.

[0041] Secondly, low-temperature methanol provides extremely rapid fixation. When the cell nucleus comes into contact with methanol at -20°C, the instantaneous cold shock effect causes proteins to rapidly denature and precipitate, thus freezing and fixing the morphology of the cell nucleus in a very short time. This rapid fixation mechanism can effectively block the activity of endogenous nucleases in the cell nucleus (such as DNase and RNase), preventing the degradation of nuclear materials in subsequent time-consuming steps such as centrifugation and washing. Especially for highly degradable macromolecules like RNA, the rapid fixation effect of pre-cooled methanol is crucial, maximizing the preservation of the integrity of RNA within the cell nucleus and ensuring the reliability of downstream transcriptome sequencing data.

[0042] Therefore, by precooling methanol to -20°C, a synergistic effect is achieved in both removing impurities and maintaining the integrity of cell nuclei, providing a strong guarantee for obtaining high-purity and high-integrity plant seed cell nuclei.

[0043] Example 3 This embodiment provides a method for extracting cell nuclei from plant fruit seeds. This method utilizes the kit described in Example 1 or 2, combining specific physical and chemical methods to achieve efficient release and purification of the cell nuclei. The method specifically includes the following steps: Step S1: Collect the seeds from the plant fruit, wash them with PBS, and then blot dry. This step aims to remove fruit pulp debris, microorganisms, and environmental impurities adhering to the seed surface to prevent them from interfering with the subsequent lysis reaction. Blotting dry is to avoid diluting the concentration of the lysis buffer added later, ensuring the stability of the lysis environment.

[0044] Step S2: Add lysis buffer to the clean seeds until completely submerged, and mechanically shear with a pre-cooled blade to create a homogenous paste of uniform size. Plant seeds typically have a hard seed coat or cell wall, which is difficult to penetrate with simple chemical lysis. This step uses a synergistic approach of mechanical shearing and chemical lysis: the pre-cooled blade rapidly breaks down the tissue structure at low temperature, physically disrupting the integrity of the cell wall; simultaneously, components such as Triton X100 in the lysis buffer quickly penetrate to the cut, chemically lysing the exposed cell membrane. This ensures effective breaking down of the hard seeds while avoiding the heat generated by excessive grinding that could lead to nuclear degradation. The degree of shearing should be such that the tissue becomes a homogenous paste of uniform size, at which point most of the cell nuclei have been released from their cell wall confinement.

[0045] Step S3: Add washing buffer to the obtained homogenate to terminate lysis, and then pipette the homogenate. When a large number of free single cell nuclei are observed in the field of view under a microscope, washing buffer should be added immediately to dilute the lysis buffer and terminate the lysis process of Triton X100, preventing nuclear membrane dissolution due to excessive lysis time. Piping the homogenate helps to disperse the cell nuclei that are stuck together, improving the quality of the mononuclear suspension.

[0046] Step S4: Collect the obtained homogenate through a cell sieve into a new centrifuge tube, centrifuge, discard the supernatant, add washing buffer to resuspend the precipitate, centrifuge again, and collect the supernatant to obtain the first coarse suspension. The cell sieve can intercept large, unbroken tissue fragments, while the filtrate contains released cell nuclei. By centrifuging at low speed, the cell nuclei precipitate at the bottom of the tube, and discarding the supernatant removes some soluble impurities.

[0047] Step S5: Collect the residue from the cell sieve, transfer it to a new sample tube, add lysis buffer, and shear with a pre-cooled blade for 5-10 minutes. After pipetting with a Pasteur pipette for 2 minutes, pass the suspension through the cell sieve, collect the suspension, centrifuge, and discard the supernatant. Add washing buffer to resuspend the precipitate, centrifuge again, and collect the supernatant to obtain the second coarse suspension. This is a key optimization step in the method of this invention. The inventors discovered that for hard, grain-like samples, a single shearing operation often fails to completely break down all tissues, leaving a large number of unreleased cell nuclei trapped in the residue. Directly discarding the residue leads to a significant decrease in the cell nucleus yield. This step performs secondary lysis and shearing on the incompletely pulverized tissue in the residue, fully releasing the remaining cell nuclei and significantly improving the final overall yield.

[0048] The centrifugation parameters in steps S4 and S5 were optimized as follows: the parameters were the same for both centrifugation steps: 4℃, 500-600 rcf, centrifugation for 5-10 min; and 4℃, 50-100 rcf, centrifugation for 1 min. This parameter combination embodies the combination of differential centrifugation and gentle separation. First, the step of centrifugation at 500-600 rcf for 5-10 min aims to precipitate and enrich cell nuclei from the large amount of liquid. This speed is sufficient to allow cell nuclei to settle, but it is not as fast as higher speeds that would tightly compact heavy impurities such as cell wall fragments and starch granules to the bottom of the tube, facilitating subsequent resuspension. Subsequently, after adding washing buffer to resuspend the precipitate, centrifugation at a very low speed of 50-100 rcf for 1 min is used, which is a crucial impurity removal step. Because cell nuclei have a relatively high specific gravity but good suspension properties, while large particles such as unbroken tissue fragments and starch granules have an even higher specific gravity and a faster settling speed, this low-speed centrifugation allows large particles of impurities to quickly settle to the bottom of the tube, while intact cell nuclei remain suspended in the supernatant. At this point, the supernatant is aspirated, which effectively removes large particulate impurities and significantly improves the purity of the crude suspension. If the rotation speed is too high or the time is too long, cell nuclei will also precipitate, leading to a decrease in yield; if the rotation speed is too low, the impurity removal effect will be insignificant.

[0049] Step S6: Combine the first and second crude suspensions to obtain a mixed suspension. This merging operation collects the products from both extractions, preparing for subsequent unified purification.

[0050] Step S7: After centrifuging the mixed suspension, discard the supernatant, add pre-cooled methanol at -20 ℃ to resuspend the precipitate, and then fix it at -20 ℃; centrifuge to enrich the precipitate, resuspend it with washing buffer, centrifuge again, wash away the methanol components, add washing buffer to the precipitate to resuspend the precipitate. This step utilizes the organic solvent properties of methanol to remove hydrophobic impurities such as lipids, and maintains the morphology of cell nuclei through low-temperature fixation.

[0051] Step S8: Add Percoll solution diluted with washing agent to the sample tube sequentially: 60% v / v Percoll solution, 25% v / v Percoll solution, and coarse cell nucleus suspension, forming a discontinuous three-layer structure; centrifuge to separate the layers. Figure 1 As shown, this is the core step in fine purification using the density gradient centrifugation principle. Percoll medium forms a continuous or discontinuous density gradient in the centrifugation field. This invention employs a discontinuous three-layer system: the bottom layer (60% v / v Percoll solution) has the highest density, the middle layer (25% v / v Percoll solution) has the second highest density, and the top layer (coarse cell nucleus suspension) has the lowest density. During centrifugation, particles of different densities move to the interface of the medium layer with equal density. Starch granules, commonly found in plant seeds, have a higher density and will pass through the middle layer to settle to the bottom layer or the bottom of the tube; while cell debris, pigments, and other impurities have a lower density and remain suspended in the upper layer or at the interface; intact cell nuclei have a density between the two, ultimately accumulating at the interface between the 25% v / v Percoll solution layer and the 60% v / v Percoll solution layer. Figure 1 The second layer is shown in the right image. This precise density positioning enables efficient separation of the cell nucleus from light and heavy impurities.

[0052] In this step, the volume ratio of the three liquids—60% v / v Percoll solution, 25% v / v Percoll solution, and coarse cell nucleus suspension—is 1:1:1. This volume ratio is crucial for forming a stable discontinuous density gradient interface. In density gradient centrifugation, the formation of a discontinuous gradient depends on a clear interface between liquid layers of different densities. If the volume of the coarse cell nucleus suspension layer is too large, its gravity may dissipate the lower 25% v / v Percoll solution interface before or in the early stages of centrifugation, leading to a blurred gradient and preventing precise separation of cell nuclei from impurities. If the volume is too small, the throughput will be insufficient. Using a 1:1:1 volume ratio ensures that the height and density difference of each liquid layer are matched, forming a clearly defined three-layer structure in the centrifuge tube. This ensures that cell nuclei can accurately migrate and accumulate at the interface between the 25% v / v Percoll solution and the 60% v / v Percoll solution—the second layer—under centrifugal force, thereby achieving effective separation from both light and heavy impurities.

[0053] In step S8, density gradient centrifugation is performed at 4 °C, 1000-2000 rcf, for 5-10 min, with a centrifuge acceleration / deceleration rate of 4°C / 4°C. Compared to conventional centrifugation, density gradient centrifugation requires greater centrifugal force to drive particle migration in the Percoll medium, which has a certain viscosity. A centrifugal force of 1000-2000 rcf is sufficient to encourage cell nuclei to overcome medium resistance and rapidly move to the interface layer with the same density. If the centrifugal force is below 1000 rcf, cell nuclei may remain in the upper or middle layers and fail to reach the target interface, resulting in low separation efficiency. If the centrifugal force exceeds 2000 rcf or the time is too long, cell nuclei may be excessively compressed and deformed, or even pass through the 60% v / v Percoll solution layer and settle to the bottom of the tube, mixing with heavy impurities and affecting the final suspension quality.

[0054] Meanwhile, the centrifuge's acceleration and deceleration speed of 4 increments followed by 4 decelerations is crucial for maintaining the stability of the discontinuous density gradient interface. During density gradient centrifugation, the centrifuge's acceleration and deceleration speeds directly affect the hydrodynamic state of the liquid. If acceleration is too rapid (e.g., accelerating to speed 7-9), the violent acceleration process will generate eddies in the liquid, disturbing the clear interface between the 25% v / v Percoll solution and the 60% v / v Percoll solution, resulting in gradient blurring and cell nuclei failing to accurately locate at the target interface. If deceleration is too rapid (e.g., decelerating to speed 7-9), the violent braking effect will generate reverse eddies, causing the separated cell nuclei to remix with impurities, destroying the separation effect. Using a moderate acceleration and deceleration speed of 4 increments followed by 4 decelerations ensures centrifugation efficiency while avoiding eddy disturbances to the gradient interface. During the acceleration phase, the liquid gradually establishes a density gradient in a stable centrifugation field, and particles migrate orderly to the target density layer; during the deceleration phase, the liquid decelerates smoothly, and the separated particles remain stable without remixing. Ultimately, cell nuclei were precisely enriched at the interface between 25% v / v Percoll solution and 60% v / v Percoll solution (the second layer), achieving efficient separation from both light and heavy impurities.

[0055] In summary, this parameter range ensures that the cell nuclei complete their stratified positioning within the optimal time, resulting in a high-purity cell nucleus suspension.

[0056] Step S9: Aspirate the liquid from the second layer and transfer it to a sample tube containing washing buffer; centrifuge to enrich the precipitate, then resuspend the precipitate in washing buffer to obtain a pure cell nuclear suspension. Aspirating only the liquid from a specific layer avoids contamination from bottom precipitate or top waste liquid. The resulting cell nuclear suspension has high purity and intact morphology, and can be directly used for downstream experiments such as single-cell sequencing.

[0057] In steps S7 and S9, the centrifugation parameters were set to 4 °C, 500-600 rcf, and 5 min. These two steps involve washing after methanol treatment and final collection after Percoll gradient centrifugation, respectively. These parameters are standard enrichment conditions optimized for the physical properties of cell nuclei. The low temperature of 4 °C inhibits enzyme activity and prevents degradation; the centrifugal force of 500-600 rcf effectively overcomes the viscosity of the liquid, especially in systems containing methanol or Percoll, precipitating the cell nuclei to the bottom of the tube while avoiding excessive centrifugation force that would cause the cell nuclei to adhere tightly to the tube wall and be difficult to resuspend, or mechanical compression that could cause the nuclear membrane to rupture. The 5 min centrifugation time is sufficient to complete the precipitation process and avoids the thermal effects or nuclear aging caused by excessively long centrifugation.

[0058] Example 4 This embodiment uses fresh grapes as a specific application to detail the process and effects of extracting cell nuclei from plant fruit seeds using the kit and method described in this invention. This application embodiment aims to verify the feasibility and superiority of the above technical solution in practical operation, ultimately obtaining a high-purity cell nucleus suspension.

[0059] The specific operating procedure is as follows: Step S1: Select a number of fresh grapes of moderate ripeness, crush them manually, and separate the seeds. Wash the seeds with 1XPBS buffer to thoroughly remove any attached pulp fragments and sugars, then blot dry with lint-free paper. Removing moisture is crucial for maintaining the accuracy of the subsequent lysis buffer concentration.

[0060] Step S2: Place the washed grape seeds in a dry petri dish and add 3 mL of lysis buffer, ensuring the seeds are completely submerged. Mechanically shear the seeds using a pre-chilled shear blade on ice for 10 minutes, until the tissue forms a homogeneous paste. This step is performed entirely on ice, utilizing the low temperature to inhibit endogenous nuclease activity. Combined with the Triton X100 component in the lysis buffer, this allows for simultaneous physical disruption of the cell wall and chemical lysis of the cell membrane.

[0061] Step S3: Add 3 mL of washing solution to the obtained homogenate to dilute the lysis buffer concentration and terminate the lysis reaction. Then, use a Pasteur pipette to repeatedly blow and agitate the homogenate, using the shear force of the liquid flow to disperse the adhering clumps of cell nuclei into individual cell nuclei, preventing subsequent filtration clogging.

[0062] Step S4: Filter the homogenized slurry through a 40 μm sieve into a 50 mL centrifuge tube. Centrifuge the filtrate at 500 rcf for 10 min at 4 ℃. After centrifugation, discard the supernatant, add 2 mL of washing buffer to the precipitate and resuspend, then centrifuge at 100 rcf for 1 min at 4 ℃. At this point, the heavier, unbroken tissue fragments and some starch granules will precipitate at the bottom of the tube. Aspirate the supernatant to obtain the first coarse suspension.

[0063] Step S5: Collect the residue retained on the cell sieve and transfer it to a new culture dish. Add 3 mL of lysis buffer again. Perform a second cut using a pre-cooled blade for 10 min, followed by pipetting with a Pasteur pipette for 4 min to extract the remaining cell nuclei. Pass the suspension through a 40 μm cell sieve again. Centrifuge the filtrate at 500 rcf for 10 min, discard the supernatant, resuspend in washing buffer, and centrifuge at 50 rcf for 1 min. Collect the supernatant to obtain the second coarse suspension. This secondary extraction step significantly improved the total yield of cell nuclei from firm grape seeds.

[0064] Step S6: Combine the first coarse suspension and the second coarse suspension to obtain a mixed suspension.

[0065] Step S7: Centrifuge the mixed suspension at 4℃ and 500 rcf for 5 min, and discard the supernatant. Add 1 mL of methanol solution pre-cooled at -20℃ to the precipitate, resuspend the precipitate, and fix it at -20℃ for 20 min. Then centrifuge at 500 rcf for 5 min to enrich the precipitate, wash once with washing buffer to remove methanol residue, and finally resuspend the precipitate with an appropriate amount of washing buffer. The crude suspension mixture of the sample after this step is as follows: Figure 4 As shown, the crude suspension of the sample appears as a white, flocculent precipitate that can be blown away.

[0066] Step S8: Construct a discontinuous density gradient system in a 15 mL centrifuge tube: Carefully add Percoll solution diluted with washing agent sequentially: 3 mL of 60% v / v Percoll solution, 3 mL of 25% v / v Percoll solution, and 3 mL of the above-mentioned crude cell nucleus suspension, with a volume ratio of 1:1:1, forming a clearly defined three-layer structure. Centrifuge at 2000 rcf for 10 min at 4 ℃. After centrifugation, liquid stratification is visible, with cell nuclei enriched in the second layer: the interface between the 25% v / v Percoll solution layer and the 60% v / v Percoll solution layer.

[0067] Step S9: Carefully aspirate the second layer of liquid using a wide-mouth pipette and transfer it to a centrifuge tube containing 2 mL of washing buffer. Add washing buffer to a final volume of 12 mL and centrifuge at 4°C and 500 rcf for 5 min to remove Percoll media. Discard the supernatant and resuspend the precipitate in 800 μL of washing buffer to obtain a pure grape seed cell nucleus suspension.

[0068] Result verification: Take 5 μL of the obtained cell nuclear suspension, mix it with an equal volume of trypan blue dye, and observe it under a microscope.

[0069] The results are as follows Figure 2 As shown, under a 20X objective lens, numerous scattered, dark blue, round or oval particles are visible; these are the stained cell nuclei. The nuclei are plump with clear, sharp edges, and no obvious cell debris or impurities are observed in the background. This indicates that the kit and method described in this embodiment successfully extracted a highly intact and pure cell nucleus suspension from fresh grape seeds. This suspension directly meets the sample quality requirements for downstream single-cell sequencing library construction.

[0070] Example 5 To further verify the effect of the concentration range of key components in the lysis buffer on the cell nucleus extraction effect, this embodiment set up a comparative experiment, focusing on the effect of Triton X100 concentration changes on cell nucleus integrity.

[0071] Fresh grape seeds from a consistent source were selected as experimental materials and divided into two groups for parallel operation. The lysis buffer formulation described in Example 1 of this invention was used, with the concentration of Triton X100 set at 0.5% w / v in one group and the concentration of Triton X100 adjusted to 1% w / v in the other group. The concentrations of other components and the operating steps (including mechanical shearing time, centrifugation parameters, purification process, etc.) were completely consistent with those in Example 4.

[0072] Experimental results refer to Figure 3 As shown in the figure, the left image shows the microscopic examination results of the first group (0.5% w / v Triton X100), and the right image shows the microscopic examination results of the second group (1% w / v Triton X100). It can be observed that the cell nuclei are severely damaged, with many cell nuclei having blurred edges and irregular shapes. Some cell nuclei have even completely disintegrated, and particulate impurities caused by the leakage of nuclear contents are scattered in the background.

[0073] Triton X100, a nonionic surfactant, works by disrupting the lipid bilayer to lyse the cell membrane. However, the nuclear membrane is also composed of a lipid bilayer. When the concentration of Triton X100 is within the suitable range of 0.04%-0.4% w / v, it is sufficient to penetrate the cell wall gaps and lyse the cell membrane to release the nucleus, but not enough to disrupt the relatively stable nuclear membrane. Once the concentration exceeds the upper limit of this range, excessive surfactant molecules will over-insert into the nuclear membrane lipid bilayer, leading to the collapse of the nuclear membrane structure, resulting in nucleus rupture and leakage of contents. This not only leads to a decrease in nucleus yield, but the leaked chromatin and RNA also adhere to impurities, severely contaminating the final suspension sample and making it unsuitable for downstream experiments such as single-cell sequencing. Therefore, this invention strictly limits the concentration of Triton X100 in the lysis buffer to 0.04%-0.4% w / v, which is a key technical means to balance sufficient release and maintenance of integrity, and has non-obvious technical benefits.

[0074] Example 6 To further verify the effect of key components in the purification solution on the cell nucleus extraction effect, a comparative experiment was set up in this embodiment, focusing on the effect of methanol on the cell nucleus extraction effect.

[0075] The concentration of reagents and operating procedures (including mechanical shearing time, centrifugation parameters, extraction process, etc.) used were completely consistent with those in Example 4. The difference was that in the purification step, the mixed suspension was centrifuged at 4°C and 500 rcf for 5 min, the supernatant was discarded, and the precipitate was resuspended with an appropriate amount of washing solution.

[0076] The effect of Percoll density gradient centrifugation is as follows Figure 5 As shown.

[0077] The results showed that there was a layer of green products in the upper layer. The green products produced in this layer gradually turned dark brown in color over time during subsequent operations. This substance may be secondary metabolites such as lipids and pigments that are abundant in plant seeds. If the removal is incomplete, the metabolites will affect subsequent sequencing.

[0078] Combined with the crude suspension of the sample treated with methanol in Example 4, such as Figure 4 As shown, it appears as a blown-away white flocculent precipitate, indicating that methanol treatment can remove most of the metabolites, thereby improving the quality of the final RNA and reducing the risk of degradation.

[0079] Example 7 To further verify the effect of key components in the purification solution on the cell nucleus extraction effect, this embodiment sets up a comparative experiment, focusing on the effect of Percoll solution concentration on the cell nucleus extraction effect.

[0080] Fresh grape seeds from a consistent source were selected as experimental materials and divided into three groups for parallel operation. The difference was that different Percoll solution combinations were used for separation: one group used only 80% v / v Percoll solution; the second group used 10% v / v Percoll solution + 50% v / v Percoll solution; and the third group used 20% v / v Percoll solution + 60% v / v Percoll solution. The concentrations of other components and the operating procedures (including mechanical shearing time, centrifugation parameters, purification process, etc.) were completely consistent with those in Example 4.

[0081] Separation results as follows Figure 6 As shown, the microscopic examination results are as follows: Figure 7 As shown.

[0082] The results showed that in the first group, most impurities could not penetrate the Percoll solution layer after centrifugation, resulting in separation failure. In the second group, microscopic examination of the interlayer revealed very few cell nuclei, with most nuclei remaining in the precipitate. In the third group, the separation was relatively good, and the microscopic examination results were also cleaner, but impurities were still present.

[0083] Example 8 To further verify the effect of key components in the purification solution on the cell nucleus extraction effect, a comparative experiment was set up in this embodiment, focusing on the effect of the centrifuge's lifting and lowering speed on the cell nucleus extraction effect.

[0084] Fresh grape seeds from the same source were selected as experimental materials and divided into two groups for parallel operation. Centrifuges were used to separate the seeds at different lifting and lowering speeds: one group lifted the seeds at a speed of 1:1 and lowered them at a speed of 9:9. The concentrations of other components and the operating procedures (including mechanical shearing time, centrifugation parameters, purification process, etc.) were completely consistent with those in Example 4.

[0085] The results showed that the first group had extremely low separation efficiency and long operation time; the second group had poor Percoll particle distribution. Figure 8 The possible reason is that during the rapid ascent and descent (9 steps), the intense eddies and reverse eddies caused interface disruption, leading to separation failure.

[0086] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for extracting cell nuclei from plant fruit seeds, characterized in that: The kit includes lysis buffer, washing buffer, and purification buffer; The lysis buffer comprises 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / μL RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS, and 200-350 mM sucrose; The cleaning solution comprises 2% w / v BSA, 1.25-2.5 mM DTT, and 0.2-0.4 U / μL of RNase inhibitor; The purification solution comprises methanol and at least two Percoll solutions of different concentrations.

2. The kit for extracting cell nuclei from plant fruit seeds according to claim 1, characterized in that: The methanol is pre-cooled at -20°C before use.

3. The kit for extracting cell nuclei from plant fruit seeds according to claim 1, characterized in that: The Percoll solution includes a Percoll solution with a concentration of 60-65% v / v and a Percoll solution with a concentration of 25-30% v / v.

4. A method for extracting cell nuclei from plant fruit seeds using the kit according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Take the seeds from the plant fruit, wash them with PBS and then dry them. S2. Add the lysis solution to the clean grains until they are completely submerged, and mechanically cut them into a homogenous slurry of uniform size using a pre-cooled blade. S3. Add the cleaning solution to the obtained homogenate to terminate the pyrolysis, and blow the homogenate with a Pasteur pipette. S4. Collect the obtained homogenate through a cell sieve into a new centrifuge tube, centrifuge, discard the supernatant, add the washing solution to resuspend the precipitate, centrifuge again, and aspirate the supernatant to obtain the first crude suspension. S5. Take the residue from the cell sieve, transfer it to a new sample tube, add the lysis buffer, cut it with a pre-cooled blade for 5-10 min, blow it with a Pasteur pipette for 2-3 min, then pass it through the cell sieve to collect the suspension. Centrifuge and discard the supernatant. Add washing buffer to resuspend the precipitate, centrifuge, and aspirate the supernatant to obtain the second coarse suspension. S6. Combine the first coarse suspension and the second coarse suspension to obtain a mixed suspension; S7. After centrifuging the mixed suspension, discard the supernatant, add methanol pre-cooled at -20℃ to resuspend the precipitate, and then place it at -20℃ for fixation; centrifuge to enrich the precipitate, resuspend it with the washing solution, centrifuge again, wash away the methanol component, and add the washing solution to the precipitate to resuspend the precipitate. S8. Add Percoll solution of different concentrations to the sample tube in descending order of concentration, then add the resuspended cell nucleus suspension to form a discontinuous liquid layer, and centrifuge. S9. Absorb the liquid from the second layer and transfer it to a sample tube containing the cleaning solution; After centrifugation and enrichment, the precipitate is resuspended in the washing solution to obtain a pure cell nucleus suspension.

5. The method for extracting cell nuclei from plant fruit seeds according to claim 4, characterized in that: In step S8, the volumes of Percoll solutions of different concentrations and crude cell nucleus suspensions added are the same.

6. The method for extracting cell nuclei from plant fruit seeds according to claim 4, characterized in that: The centrifugation parameters in steps S4 and S5 are the same: 4 ℃, 500-600 rcf, centrifugation for 5-10 min; 4 ℃, 50-100 rcf, centrifugation for 1 min.

7. The method for extracting cell nuclei from plant fruit seeds according to claim 4, characterized in that: In steps S7 and S9, the centrifugation parameters are 4 ℃, 500-600 rcf, and centrifugation for 5 min.

8. The method for extracting cell nuclei from plant fruit seeds according to claim 4, characterized in that: In step S8, the centrifugation parameters are 4 ℃, 1000-2000 rcf, and 5-10 min.

9. The method for extracting cell nuclei from plant fruit seeds according to claim 8, characterized in that: The centrifuge speed during the centrifugation process is 4 increments and 4 decrements.

10. A suspension of plant fruit seed cell nuclei, characterized in that: It is prepared by the method described in any one of claims 4-9.

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