Method for separating cotton boll cell nucleuses in dehydration mature period

Abstract

The invention provides a method for separating cotton boll cell nucleuses in a dehydration mature period, which comprises the following steps: cotton boll tissues in the dehydration mature period are taken and ground into fine powder under the protection of liquid nitrogen, and the tissues must comprise seed coat parts; filtering the first separation liquid through a first aperture filter, and collecting filtrate; carrying out low-temperature centrifugation on the filtrate, discarding the supernatant, and adding a second separation liquid containing a nonionic detergent to resuspend and precipitate; performing low-temperature centrifugation on the re-suspended precipitate again, discarding the supernatant, and washing the precipitate for multiple times by using a high-salt washing solution; resuspending the washed precipitate by using a third separation liquid containing a higher osmotic pressure component; after centrifugation is finished, a cell nucleus enrichment interface layer is sucked; diluting the interface layer solution with a third separating solution, and then filtering again through a second aperture filter; and carrying out low-temperature centrifugal collection on the filtered solution to obtain separated cotton boll cell nucleuses.

Description

Technical Field

[0001] This invention relates to the field of information technology, and in particular to a method for separating the nuclei of dehydrated, mature cotton boll cells. Background Technology

[0002] Cotton genetics and molecular biology research is a core area of ​​agricultural science, crucial for improving cotton quality, yield, and stress resistance. This research not only relates to the sustainable development of agricultural production but also directly impacts the raw material supply for the textile industry, thus attracting significant global attention. In-depth research into the genetic information of cotton cells can provide important evidence for breeding and gene improvement.

[0003] However, current methods for extracting cotton cell nuclei face numerous challenges, especially when processing dehydrated, mature cotton boll tissue, where the results are often unsatisfactory. Many traditional techniques struggle to address the high stiffness and fiber content of cotton boll tissue, easily damaging the integrity of cell nuclei during operation. Furthermore, the extracted nuclei often have low purity, failing to meet the needs of subsequent research. These problems frequently limit researchers' access to reliable experimental materials.

[0004] At a deeper level, the extraction of cell nuclei from cotton boll tissue faces a key technical challenge: ensuring the complete release and effective separation of the nuclei while avoiding mechanical damage. During the dehydration and maturation stage, cotton boll tissue exhibits a very tough structure with tightly intertwined internal fibers. If conventional grinding or separation methods are used directly, the cell nuclei are easily ruptured due to external pressure. Worse still, although the seed coat contains abundant cell nuclei, their unique tissue characteristics mean they are firmly encased and difficult to release gently. This situation results in insufficient quantity or poor quality of extracted cell nuclei, even with significant time and effort.

[0005] In practical applications, such as grinding cotton boll tissue, excessive force can directly damage the cell nuclei, while insufficient force will fail to break down the tissue, preventing the nuclei from being separated from the seed coat. Furthermore, even if some nuclei are managed to be released, subsequent separation steps are extremely difficult due to interference from fibrous impurities and cell debris, resulting in nuclei that are often mixed with other substances and unsuitable for precise genetic analysis.

[0006] Therefore, how to efficiently release and purify high-quality cell nuclei from the tough, high-fiber dehydrated mature cotton boll tissue while protecting the integrity of the cell nucleus has become a key problem that needs to be solved in current research. Summary of the Invention

[0007] This invention provides a method for separating cell nuclei from dehydrated, mature cotton bolls, mainly comprising: Dehydrated, mature cotton boll tissue was collected and ground into a fine powder under liquid nitrogen protection. The tissue must include the seed coat. The fine powder was added to a pre-cooled first separation solution containing specific components, followed by brief homogenization and ice bath settling. The first separation solution was filtered through a first-pore filter, and the filtrate was collected. The filtrate was centrifuged at low temperature, the supernatant was discarded, and the precipitate was resuspended in a second separation solution containing a non-ionic detergent. The resuspended precipitate was centrifuged again at low temperature, the supernatant was discarded, and the precipitate was washed multiple times with a high-salt washing solution. The washed precipitate was resuspended in a third separation solution containing a high-osmotic-pressure component. The resuspended solution was stacked on two pre-laid Percoll density gradient solutions of different concentrations and subjected to density gradient centrifugation. After centrifugation, the nucleus-enriched interface layer was aspirated. The interface layer solution was diluted with the third separation solution and filtered again through a second-pore filter. The filtered solution was centrifuged at low temperature to collect the separated cotton boll cell nuclei. Furthermore, the step of taking dehydrated mature cotton boll tissue and grinding it into a fine powder under liquid nitrogen protection, wherein the tissue must contain the seed coat portion, includes: controlling the weight of the selected cotton boll tissue to a very small range; ensuring that the selected tissue contains the seed coat portion during sampling; and rapidly grinding the cotton boll tissue containing the seed coat in liquid nitrogen until a uniform fine powder is formed. Further, the step of filtering the first separation liquid through a first-pore size filter and collecting the filtrate includes: using a first filter with a pore size of 40 μm to perform a first coarse filtration of the separation liquid containing the initially dispersed tissue; the first filter is used to retain most of the large cotton fiber particles and insufficiently broken tissue fragments; collecting the filtrate after passing through the first filter as the starting material for subsequent processing. Further, the step of adding a second separation liquid containing a non-ionic detergent to resuspend the precipitate includes: adding Triton X-100 to the second separation liquid in addition to the components of the first separation liquid; controlling the concentration of Triton X-100 at a low level to achieve gentle lysis; and using a slow blowing method to fully mix the precipitate with the second separation liquid. Furthermore, the step of stacking the resuspension onto two pre-laid Percoll density gradient solutions of different concentrations for density gradient centrifugation includes: pre-laying two solutions of different Percoll concentrations sequentially at the bottom of the centrifuge tube to form a discontinuous density gradient; the first layer of Percoll solution contains a higher proportion of Percoll components; the second layer of Percoll solution contains the highest proportion of Percoll components; carefully stacking the resuspension containing cell nuclei onto the top layer of the density gradient; and performing a relatively long period of medium speed and low temperature centrifugation to effectively separate the different density components in the gradient.Furthermore, the process of aspirating the nuclear enrichment interface layer and subsequent treatment after centrifugation includes: observing a distinct nuclear enrichment band at the interface between the two Percoll density layers after centrifugation; carefully aspirating the nuclear enrichment band into a new container; diluting the aspirated nuclear enrichment solution with a third separation buffer; and finely filtering the diluted solution using a second filter with a smaller pore size (20 μm). Furthermore, the common components of the first separation buffer, the second separation buffer, and the washing buffer include: all containing a Tris-HCl buffer system; and all containing spermidine; the spermidine is used to stabilize the nuclear structure.

[0008] The technical solutions provided by the embodiments of the present invention may include the following beneficial effects: This invention discloses a method for separating cell nuclei from dehydrated mature cotton bolls. Addressing the unique challenges of high rigidity, high fiber content, and easily damaged cell nuclei due to mechanical and enzymatic degradation in dehydrated mature cotton boll tissue, as well as the difficulty in extracting intact nuclei from the abundant seed coat portion, this method employs a series of precisely integrated cryogenic protection and multi-stage purification techniques. First, the cotton boll tissue, including the seed coat, is ground into a fine powder under liquid nitrogen to avoid mechanical damage and enzyme activity degradation. Then, a pre-cooled first separation solution with optimized components is used to gently release the cell nuclei, followed by filtration through a 40-micron pore size to remove large fibrous impurities. The precipitate is then collected by low-speed centrifugation and resuspended in a second separation solution containing 0.1% to 0.5% Triton X-100 to remove cytoplasm and membrane fragments. Next, multiple washes with a high-salt washing solution are performed to remove residual contaminants. Finally, after resuspending in a hypertonic third separation solution, a 30%-80% Percoll discontinuous density gradient centrifugation is performed to precisely enrich the cell nuclei at the interface layer. A second 20-micron filtration and final centrifugation yield high-purity, intact cell nuclei. This invention effectively solves the core problems of severe cell nucleus damage, low purity, and poor representativeness in traditional methods, providing an efficient, stable, and reproducible technical means for cotton genetics and molecular biology research, and has significant practical value and prospects for promotion. Attached Figure Description

[0009] Figure 1 This is a flowchart of a method for separating cell nuclei from dehydrated, mature cotton bolls according to the present invention; Figure 2 This is a microscopic image of cotton boll cell nuclei from an embodiment of the present invention; Figure 3 This is a quality control image of the ATAC library of cotton boll cell nuclei, as shown in an embodiment of the present invention. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0011] like Figure 1 The method for separating cell nuclei from dehydrated, mature cotton bolls in this embodiment may specifically include: This invention provides a method for isolating cell nuclei from dehydrated, mature cotton bolls, aiming to provide an efficient and stable technical means for research in related fields. The technical solution of this invention is described in detail below with reference to specific embodiments to make the purpose, technical solution, and advantages of this invention clearer. In one embodiment, the method provided by this invention mainly targets dehydrated, mature cotton boll tissue. Through a series of meticulous separation and purification steps, high-quality cell nuclei are extracted from complex plant tissue, laying the foundation for subsequent genetic research or molecular biological analysis. This method includes several key steps, from tissue sampling to the final collection of cell nuclei, each step of which is optimized to ensure the integrity and purity of the cell nuclei. The specific operation and precautions of each step will be described step by step according to the logical order of technical implementation. Step S1: Take dehydrated, mature cotton boll tissue and grind it into a fine powder under liquid nitrogen protection. The tissue must include the seed coat. Specifically, dehydrated, mature cotton boll tissue is usually in a state of low physiological activity, with a relatively stable cell structure, but high tissue hardness. Direct processing can easily lead to cell nucleus damage. Therefore, during sampling and grinding, low-temperature protection measures are required to reduce mechanical damage and enzymatic hydrolysis. The use of liquid nitrogen can rapidly lower the tissue temperature, inhibit the activity of endogenous enzymes, and simultaneously harden the tissue, making it easier to grind into a fine powder. It should be noted that the seed coat is an important component of the cotton boll tissue, containing abundant cell nuclei. The absence of the seed coat may result in insufficient or unrepresentative cell nuclei extraction. In one possible implementation, step S1 can be further refined into the following sub-steps.

[0012] Step S11: Select cotton boll tissue at the dehydrated maturity stage, ensuring the tissue is completely dehydrated, typically characterized by a dry boll shell and white or light yellow fibers. Prioritize bolls free from obvious pests, diseases, and mechanical damage to avoid interference from impurities or degradation products.

[0013] Step S12: Place the selected cotton boll tissue in a pre-cooled mortar. The mortar and pestle need to be cooled to near liquid nitrogen temperature in a low-temperature environment in advance to prevent the tissue from degrading due to temperature rise in the early stage of grinding.

[0014] Step S13: Slowly pour liquid nitrogen into the mortar, ensuring the nitrogen covers the tissue surface. Then, use a pestle to grind rapidly for 2 to 3 minutes until the tissue is completely transformed into a uniform fine powder. During grinding, continuously replenish liquid nitrogen to maintain the low-temperature environment and prevent damage to the cell nucleus structure due to frictional heating. For example, in practice, researchers can collect mature cotton bolls from the field, prioritizing bolls that have naturally split open in the later stages of the planting cycle. When sampling, carefully cut the cotton bolls with scissors to avoid tearing the tissue. Then, quickly place the sample in a portable cryogenic chamber filled with liquid nitrogen and transport it to the laboratory. In the laboratory, the cotton boll tissue is placed in the mortar in batches, with each batch controlled at 1 to 2 grams to ensure uniform grinding. During grinding, the operator must wear anti-freeze gloves and goggles to avoid injury from liquid nitrogen splashes. This method effectively protects the integrity of the cell nucleus, providing high-quality starting material for subsequent separation steps. In another embodiment, the tissue hardness and fiber content may differ for different varieties of cotton boll tissue, such as long-staple cotton and short-staple cotton. Therefore, the grinding time and liquid nitrogen dosage need to be adjusted appropriately. For long-staple cotton with a higher fiber content, the grinding time can be extended to 3 to 4 minutes, while increasing the frequency of liquid nitrogen replenishment to avoid uneven grinding caused by fiber entanglement in the grinding pestle. For short-staple cotton with fewer fibers, the grinding time can be appropriately shortened to 1.5 to 2 minutes to reduce the potential damage to cell nuclei caused by excessive grinding. This flexible adjustment method can adapt to the characteristics of different cotton boll samples and ensure the consistency of grinding effect. In step S2, the fine powdered tissue is added to a pre-cooled first separation solution containing specific components, followed by a brief homogenization and ice bath standing treatment. Specifically, the first separation solution is an optimized buffer solution whose main function is to initially disperse the tissue powder, release the cell nuclei, and protect the cell nuclei from mechanical stress and osmotic pressure changes through specific components. Pre-cooling further reduces the solution temperature and enzyme activity, while brief homogenization helps the tissue powder and separation solution to come into full contact, and ice bath incubation provides a mild environment for the initial release of cell nuclei. In one embodiment, step S2 can be broken down into the following sub-steps.

[0015] Step S21: Prepare the first separation solution. The separation solution needs to be pre-cooled at 4 degrees Celsius for at least 1 hour in advance. Its components include sugars to maintain osmotic pressure, buffers to stabilize cell nuclear structure, and protectants to inhibit protease activity.

[0016] In step S22, the fine powder obtained from grinding is slowly added to the pre-cooled first separation liquid. The ratio of tissue to separation liquid is controlled between 1:5 and 1:10 to ensure that the tissue is fully dispersed.

[0017] Step S23: Use a homogenizer to briefly homogenize the mixture, controlling the homogenization time to be between 30 and 60 seconds, and setting the speed to medium to low to avoid over-homogenization that could cause cell nuclei to rupture.

[0018] Step S24: Place the homogenized mixture in an ice bath and let it stand for 10 to 15 minutes, gently shaking the container during this time to promote the release of cell nuclei. For example, in laboratory procedures, 1 gram of finely powdered cotton boll tissue can be added to 10 ml of pre-cooled first separation solution, which is pre-placed in an ice box to maintain a low temperature. During homogenization, use a handheld homogenizer set to 5000 rpm, strictly controlling the homogenization time to 40 seconds. Immediately afterward, transfer the mixture to a container filled with crushed ice and let it stand. During the standing process, gently shake the container every 3 minutes to ensure sufficient contact between the tissue particles and the separation solution. This method releases cell nuclei under gentle conditions while avoiding damage to cell nuclei due to excessive mechanical force. In another possible implementation, the volume of the first separation solution and the amount of tissue added can be adjusted appropriately according to the experimental needs of different scales. For example, in small-scale experiments, 0.5 grams of tissue can be added to 5 ml of separation solution, and the homogenization time can be shortened to 30 seconds to reduce the operation time. In larger-scale nuclear extraction, 2 grams of tissue can be added to 20 ml of separation solution, the homogenization time extended to 60 seconds, and the ice bath incubation time increased to 20 minutes to ensure full release of the nuclei. This flexibility adapts to the needs of different experimental scenarios and improves the operability of the method. It should be noted that the design of the components of the first separation solution is one of the key innovations of this method; the choice of its buffer system and protective agent directly affects the integrity of the nucleus. For example, spermidine added to the separation solution can bind to nucleic acids in the nucleus, forming a stable complex, thereby protecting the structural stability of the nucleus during subsequent separation processes. Furthermore, the sugars in the separation solution can regulate the osmotic pressure of the solution, preventing the nucleus from swelling or shrinking due to osmotic imbalance. Through this component optimization, the nucleus is effectively protected in the initial separation stage, laying the foundation for subsequent steps. In one embodiment, the component ratio of the first separation solution can be fine-tuned to meet the experimental needs under different environmental conditions. For example, in a high-temperature and high-humidity environment, the concentration of the protective agent in the separation solution can be appropriately increased to enhance the inhibitory effect on enzyme activity. In a low-temperature, dry environment, the concentration of sugars can be appropriately reduced to avoid excessively high solution viscosity affecting the homogenization effect. This targeted adjustment ensures the stability of the method under different experimental conditions while improving the success rate of cell nucleus extraction. Step S3 involves filtering the first separation solution through a first-pore size filter and collecting the filtrate. Specifically, the purpose of the filtration step is to remove large particulate impurities from the homogenized mixture, such as incompletely broken cotton fibers, seed coat fragments, and other tissue residues, thereby providing a relatively pure starting material for subsequent cell nucleus separation. The selection of the first-pore size filter needs to balance filtration efficiency and cell nucleus throughput, avoiding situations where the pore size is too small, resulting in cell nucleus retention, or too large, resulting in impurity residue. In one possible implementation, step S3 can be further broken down into the following sub-steps.

[0019] Step S31: Select a first filter with a pore size of 40 micrometers. This pore size can effectively trap most large cotton fibers and insufficiently broken tissue fragments, while allowing cell nuclei to pass through smoothly.

[0020] Step S32: Slowly pour the homogenized and settled first separated liquid into the filter. Sterile gauze or filter paper can be used to assist in the filtration process to prevent liquid from overflowing.

[0021] Step S33: Collect the filtrate after passing through the first filter. The filtrate must be immediately placed in an ice bath to prevent cell nuclei from degrading at room temperature. For example, in practice, the first separation solution can be poured into a funnel pre-installed with a 40-micron pore size nylon mesh, with a pre-cooled 50 ml centrifuge tube placed below the funnel for collecting the filtrate. During filtration, the operator should slowly pour the mixture to avoid excessive flow rate that could clog the filter mesh. If too many impurities are found on the filter mesh surface, they can be gently removed with sterile tweezers or replaced with a new filter mesh. The collected filtrate is light yellow or colorless and transparent, indicating that most large particulate impurities have been removed, providing relatively pure material for subsequent low-temperature centrifugation. In another embodiment, the filter pore size and filtration method can be adjusted appropriately according to the characteristics of different cotton boll tissues. For example, for cotton boll samples with high fiber content, a coarse filter with a 100-micron pore size can be used for preliminary filtration before the 40-micron pore size filter to reduce the risk of clogging of the fine-pore filter. For samples with looser tissue, a 40-micron pore size filter can be used directly, with the filtration time appropriately extended to improve filtrate purity. This staged filtration method effectively addresses the characteristics of different samples and improves filtration efficiency. It should be noted that the filtration step plays a crucial role in the entire nucleus separation process, directly affecting the purity and efficiency of subsequent centrifugation. By optimizing the filter pore size and operation, impurities can be removed while maximizing the retention of nuclei, avoiding poor density gradient centrifugation results due to impurity interference. Furthermore, preserving the filtrate in an ice bath further protects the integrity of the nuclei, providing stable experimental material for subsequent steps. In one embodiment, for large-scale nucleus extraction, a multi-layer filtration system can be used to further improve filtration efficiency. For example, a coarse filter with a 100-micron pore size is used in the first layer to remove large particles, followed by a transition filter with a 60-micron pore size to remove medium-sized particles, and finally a fine filter with a 40-micron pore size for final filtration. After each filtration layer, the clarity of the filtrate should be checked. If the filtrate still contains a large number of suspended particles, filtration can be repeated or the filter replaced. This multi-layer filtration method significantly improves the purity of the filtrate, creating better conditions for subsequent separation steps. For example, in field experiments or resource-limited environments where professional filtration equipment is not immediately available, multiple layers of sterile gauze can be used as a temporary filter. Specifically, four to six layers of sterile gauze are stacked in a funnel, and the first separation solution is slowly poured in. During filtration, the accumulation of impurities on the gauze surface must be continuously observed, and the gauze replaced promptly to avoid clogging. Although this method has lower filtration efficiency, it can achieve preliminary impurity removal in emergency situations, ensuring the continuity of the experiment. In another possible implementation, for any remaining minute impurities in the filtrate, a low-speed centrifugation step can be added after filtration to further clarify the filtrate.For example, the collected filtrate is placed in a centrifuge at 4 degrees Celsius and centrifuged at 1000 rpm for 3 minutes. The supernatant is then carefully aspirated for subsequent processing. This auxiliary centrifugation step can further remove suspended particles and improve filtrate quality without affecting the integrity of cell nuclei. It should be noted that the innovation of this filtration step lies in balancing impurity removal and cell nucleus retention through the rational selection of pore size and filtration method. In traditional methods, the selection of pore size is often too broad, leading to impurity residue or cell nucleus loss. This method, by optimizing the pore size and operating procedure, significantly improves the purity of the filtrate, laying a solid foundation for subsequent cell nucleus separation. This refined operation can reduce the impact of impurities on subsequent density gradient centrifugation while ensuring the integrity of cell nuclei. In one embodiment, the collection method and storage conditions of the filtrate can also be adjusted for different experimental purposes. For example, in experiments requiring immediate subsequent separation, the filtrate can be directly stored in an ice bath and proceeded to the next step as soon as possible. In scenarios requiring long-term preservation of the filtrate, a small amount of preservative can be added, and the filtrate can be aliquoted into multiple small-volume centrifuge tubes and stored in an ultra-low temperature freezer at -80 degrees Celsius. This method effectively extends the storage time of the filtrate while avoiding damage to cell nuclei from repeated freeze-thaw cycles. For example, in actual research, if the experimental site is far from the laboratory, preliminary filtration can be completed in the field, and the filtrate can be placed in a pre-cooled thermos flask with crushed ice added to maintain the low temperature, and then transported to the laboratory as soon as possible for further processing. This temporary preservation method can maximize the protection of cell nucleus activity in the filtrate under limited resources, providing reliable material support for subsequent experiments. In another implementation, for the small amount of suspended particles that may be present in the filtrate, a microscopic observation step can be added after filtration to assess the purity of the filtrate. For example, a small amount of filtrate can be dropped onto a glass slide and observed under an optical microscope. If a large number of large particles or fiber residues are found, the filtration operation can be repeated. This direct detection method can promptly identify filtrate quality problems, ensuring the smooth progress of subsequent separation steps. It should be noted that the filtration operation in step S3 is not merely a simple impurity removal step, but a crucial step in quality control throughout the entire cell nucleus separation process. By optimizing the filter pore size, filtration method, and filtrate storage conditions, the purity and efficiency of cell nucleus extraction can be significantly improved, providing high-quality starting material for subsequent low-temperature centrifugation and density gradient separation. This meticulous operational design demonstrates the innovation and practicality of this method in terms of technical implementation. In one possible implementation, the filter material and structure can also be adjusted according to the characteristics of different cotton boll tissues. For example, for samples with high fiber content, a highly abrasion-resistant nylon mesh can be used as the filter material to prevent fibers from puncturing the filter and causing impurity leakage.For samples with relatively moist tissue, a polypropylene filter with poor water absorption can be used to reduce liquid loss due to residue on the filter. This targeted selection can further improve the filtration effect and ensure the quality of the filtrate. For example, in laboratory operations, if the filtrate flow rate is found to be too slow during filtration, the tilt angle of the filter can be appropriately increased, or a negative pressure filtration device can be used to assist filtration. In specific operation, the filter is connected to the filtration flask, and a water pump generates negative pressure to accelerate the speed at which the liquid passes through the filter. It should be noted that the negative pressure should not be too strong to avoid damaging the cell nuclei due to excessive flow. This auxiliary method can significantly shorten the filtration time and improve experimental efficiency while ensuring the integrity of the cell nuclei. In another embodiment, for trace impurities that may be present in the filtrate, a magnetic stirring step can be added after filtration to further disperse suspended particles. For example, the filtrate is placed on a magnetic stirrer and stirred at a low speed of 200 revolutions per minute for 5 minutes, then allowed to stand and observe. If the suspended particles are found to have decreased, the subsequent steps can be directly performed. This gentle stirring method can improve the homogeneity of the filtrate without damaging the cell nucleus, providing better conditions for subsequent separation. This invention provides a method for separating the nuclei of dehydrated, mature cotton boll cells. The specific implementation of the subsequent steps is described in detail below. Step S4: After low-temperature centrifugation of the filtrate, the supernatant is discarded, and a second separation solution containing a non-ionic detergent is added to resuspend the precipitate. Specifically, the filtrate after filtration through the first pore size still contains a large amount of cell debris, cytoplasmic components, and small particulate impurities. Low-temperature centrifugation allows the relatively dense cell nuclei to settle to the bottom, while most of the lighter impurities remain suspended in the supernatant. The precipitate obtained after discarding the supernatant is mainly rich in cell nuclei, but further removal of residual cell membrane debris and cytoplasmic proteins is still necessary. At this point, the introduction of a second separation solution containing a non-ionic detergent for resuspension treatment can gently dissolve and disperse the membrane structure attached to the surface of the cell nucleus, while preserving the integrity of the cell nucleus as much as possible. In one embodiment, step S4 can be further subdivided into the following sub-steps.

[0022] Step S41: Transfer the collected filtrate to a pre-cooled centrifuge tube and place it in a 4-degree Celsius low-temperature centrifuge. Centrifuge at 3000 to 5000 revolutions per minute for 8 to 12 minutes to allow the cell nuclei to settle effectively.

[0023] Step S42: Carefully discard the supernatant, being careful not to disturb the sediment at the bottom.

[0024] Step S43: Prepare the second separation liquid, which is made by adding 0.1% to 0.5% Triton X-100 as a non-ionic detergent to the basic components of the first separation liquid, and pre-cool the solution to 4 degrees Celsius.

[0025] Step S44: Add an appropriate amount of second separation liquid to the precipitate. The volume ratio of precipitate to second separation liquid is usually controlled between 1:3 and 1:8.

[0026] Step S45: Resuspend the precipitate repeatedly by gently blowing and resuspending it with a pipette until it is completely and evenly dispersed, avoiding excessive air bubbles. For example, in practice, approximately 30 ml of filtrate can be aliquoted into two 50 ml centrifuge tubes and centrifuged at 4°C and 4000 rpm for 10 minutes. After centrifugation, a small amount of white to light yellow precipitate will be visible at the bottom of the tube. After discarding the supernatant, add approximately 5 ml of pre-cooled second separation solution to each tube of precipitate and gently blow and resuspend it about 20 to 30 times with a 1 ml pipette tip until the precipitate is completely dispersed into a homogeneous suspension. This gentle resuspension method effectively removes cytoplasmic residues attached to the cell nucleus surface while avoiding cell nucleus rupture caused by strong mechanical force. In another possible implementation, to address the significant differences in fiber residue levels between different batches of cotton boll tissue, the concentration of Triton X-100 and the resuspension time can be adjusted accordingly. For samples with high fiber content and high cytoplasmic viscosity, the Triton X-100 concentration can be increased to 0.4% to 0.5%, and the number of resuspension blows can be increased to 40 to 50 times to enhance the dissolution of membrane substances. For samples with low fiber content and relatively clean tissue, the concentration can be reduced to 0.1% to 0.2%, and the number of blows reduced to 15 to 20 times to prevent excessive decontamination and damage to the nuclear membrane. This strategy of flexibly adjusting the detergent concentration and treatment intensity according to sample characteristics helps to obtain relatively intact cell nuclei in different cotton boll materials. It should be noted that the concentration control of the non-ionic detergent Triton X-100 in this step is one of the key technical points. Too high a concentration can easily lead to partial dissolution or even rupture of the nuclear membrane, while too low a concentration cannot effectively remove attached cytoplasm and membrane debris. Extensive experimental comparisons have shown that the concentration range of 0.1% to 0.5% can achieve a good balance in most dehydrated mature cotton boll samples, removing most non-nuclear components while preserving the structural integrity of the cell nucleus to the greatest extent. Step S5 involves re-centrifuging the resuspended precipitate at low temperature, discarding the supernatant, and then washing the precipitate multiple times with a high-salt washing buffer. Specifically, after treatment with the second separation solution, a small amount of detergent molecules and dissolved membrane fragments may still remain on the surface of the cell nucleus. The high-salt washing buffer utilizes its high ionic strength to disrupt the weak interactions between these residual components and the cell nucleus, thereby achieving further purification. Multiple washes gradually reduce the concentration of residual contaminants, resulting in a stepwise increase in cell nucleus purity. In one embodiment, step S5 specifically includes the following steps.

[0027] Step S51: Place the resuspended and homogenized second separation liquid suspension in a centrifuge at 4 degrees Celsius and centrifuge at a speed of 5000 to 8000 revolutions per minute for 8 to 12 minutes, and collect the bottom precipitate.

[0028] In step S52, after discarding the supernatant, a pre-cooled high-salt washing solution is added. This washing solution typically contains 0.4 to 0.8 mol / L of NaCl or KCl, and the buffer system is consistent with the aforementioned separation solution.

[0029] Step S53: Gently blow and resuspend the precipitate, washing volume is generally 5 to 10 times the precipitate volume.

[0030] Step S54 involves repeating the centrifugation, supernatant discarding, and resuspension washing process at least 2 to 4 times, with slightly increased centrifugation conditions each time to gradually improve washing efficiency. For example, in a typical experimental procedure, the resuspension can be centrifuged at 6000 rpm for 10 minutes, the supernatant discarded, and 8 mL of a high-salt washing solution containing 0.5 mol / L NaCl added. The precipitate is gently agitated 30 times to resuspend it, followed by another centrifugation at 7000 rpm for 10 minutes. This washing process is repeated 3 times. The precipitate obtained after the third wash is usually whiter and denser, indicating that most soluble contaminants have been effectively removed. In another embodiment, for cotton boll samples that are particularly difficult to purify, such as those exposed to the field for a long time or slightly moldy but still usable samples, the number of washes can be increased to 4 to 5. A further enhanced wash can be performed after the third wash, using a high-salt washing solution containing 0.1% Triton X-100 to treat stubbornly attached membrane fragments. This graded enhanced washing strategy can further improve the purity of the final product while maintaining the basic integrity of the cell nucleus morphology. Step S6 involves resuspending the washed precipitate in a third separation buffer containing a component with higher osmotic pressure. Specifically, after multiple high-salt washes, the cell nuclei have lost most of their cytoplasm and membrane contaminants, but may still contain a small amount of adherent fibrous debris or aggregates. Resuspending in a third separation buffer with a higher osmotic pressure allows the cell nuclei to shrink appropriately, reducing aggregation tendency, and provides a suitable buoyancy environment for subsequent density gradient centrifugation. In one possible implementation, the third separation buffer typically contains a high concentration of sucrose or sorbitol, typically ranging from 0.8 to 1.5 mol / L, with the buffer system consistent with the aforementioned solution. During resuspension, the volume ratio of precipitate to third separation buffer is generally controlled between 1:4 and 1:10, and the precipitate is slowly pipetted approximately 20 to 40 times until a homogeneous, slightly viscous suspension is obtained. For example, 6 mL of a third separation buffer containing 1.0 mol / L sucrose can be added to the precipitate after three washes, and the precipitate can be slowly pipetted approximately 30 times to completely disperse the precipitate. The suspension is typically milky white in appearance, with moderate fluidity, making it suitable for direct superposition onto a density gradient. Step S7 involves superimposing the resuspended solution onto two pre-laid Percoll density gradient solutions of different concentrations, followed by density gradient centrifugation. Specifically, Percoll is a colloidal silica particle coated with polyvinylpyrrolidone, possessing good biocompatibility and low osmotic pressure characteristics, and is a commonly used density medium in plant cell nucleus separation. Using two discontinuous density gradients of different concentrations can form a distinct interface, causing cell nuclei to accumulate at a specific density interface during centrifugation. In one embodiment, step S7 can be further broken down into the following sub-steps.

[0031] Step S71: Prepare two Percoll working solutions in advance, for example, use a solution containing 70% to 85% Percoll for the bottom layer and a solution containing 30% to 45% Percoll for the upper layer, and dilute them appropriately with a third separation solution and adjust them to the same osmotic pressure.

[0032] Step S72: In a 15 mL or 50 mL ultracentrifuge tube, first add 5 to 8 mL of a higher concentration Percoll solution as the bottom layer, and then carefully stack 5 to 8 mL of a lower concentration Percoll solution on top of it to form a clear interface.

[0033] Step S73: Carefully spread the cell nucleus suspension resuspended in the third separation solution on the top layer of the gradient, generally adding 2 to 5 ml.

[0034] Step S74: Place the centrifuge tube in a pre-cooled horizontal rotor centrifuge at 4°C and centrifuge at a moderate speed of 8000 to 12000 rpm for 30 to 60 minutes. For example, in a common experimental protocol, add 6 ml of 80% Percoll solution to the bottom of a 15 ml centrifuge tube, then slowly add 6 ml of 40% Percoll solution on top to form a clear interface. Then carefully add approximately 3 ml of cell nucleus resuspension to the top layer and centrifuge at 10000 rpm at 4°C for 45 minutes. After centrifugation, the cell nuclei will typically form a clear white band at the 40% and 80% Percoll interface. In another possible implementation, the Percoll concentration can be adjusted for different cotton varieties or cotton bolls at different maturity levels. For example, for upland cotton with abundant fibers and a high nucleus density, the Percoll concentration in the bottom layer can be increased to 82% to 85%, and the concentration in the top layer to 42% to 45%, to better match the sedimentation characteristics of the nuclei. For Sea Island cotton with a relatively low nucleus density, the concentrations in both layers can be appropriately reduced to approximately 75% and 38%. This method of optimizing the density gradient based on the biological characteristics of the cotton species can significantly improve the enrichment efficiency of nuclei at the interface. Step S8: After centrifugation, the nucleus-enriched interface layer is aspirated. Specifically, after density gradient centrifugation, due to their unique size and density, the nuclei typically remain stably at the interface between the two Percoll concentrations, forming a visible white or milky-white band. This interface layer is the enriched nucleus component and needs to be carefully aspirated to avoid contamination with impurities from the upper or lower layers. In one embodiment, a 200 to 1000 μL pipette can be used, under good lighting conditions, to slowly aspirate the white band of the interface layer from the side of the tube, minimizing the aspiration of the upper supernatant or the lower high-density medium. Typically, 0.8 to 1.5 mL of the interfacial layer material can be aspirated at a time, depending on the gradient tube size and sample volume. For example, in a 15 mL centrifuge tube, the interfacial layer after centrifugation is usually located in the lower-upper part of the tube, with a width of about 2 to 4 mm. The operator can place the pipette tip in the center of the interfacial zone and slowly aspirate about 1.2 mL of the white material, then immediately transfer it to a new pre-cooled centrifuge tube. In step S9, the interfacial layer solution is diluted with a third separation solution and then filtered again through a second-pore filter. Specifically, the directly collected interfacial layer may still contain a small amount of Percoll particles and very fine residual fibers. Diluting it with the third separation solution reduces the viscosity of the medium, facilitating subsequent filtration; while using a second filter with a smaller pore size can further remove residual fine impurities and improve the final cell nucleus purity. In one possible implementation, step S9 includes the following steps.

[0035] Step S91: Add the aspirated interfacial layer solution to 3 to 5 times the volume of the pre-cooled third separation liquid, and gently mix and dilute.

[0036] Step S92: Select a second filter with a pore size of 20 micrometers, typically a nylon mesh or polycarbonate membrane filter.

[0037] Step S93: Slowly pass the diluted solution through the second filter and collect the filtrate.

[0038] Step S94: If necessary, rinse the filter surface with a small amount of the third separation solution and combine all the filtrates. For example, add 1.2 mL of the interface layer material to 4 mL of the third separation solution, mix gently, and then slowly push the mixture through a 20-micron nylon filter pre-installed on a syringe to collect approximately 5 mL of filtrate. This filtrate is typically uniformly milky white with almost no visible particles. Step S10: Collect the separated cotton boll cell nuclei by low-temperature centrifugation of the filtered solution. Specifically, the cell nuclei in the final filtered solution are highly purified, and the final low-temperature centrifugation allows for effective sedimentation of the cell nuclei. After discarding the supernatant, the final product is obtained. In one embodiment, the filtered solution can be placed in a 4°C centrifuge at 8000 to 12000 rpm for 10 to 15 minutes. Carefully discard the supernatant, retaining the white precipitate at the bottom, which is the separated dehydrated mature cotton boll cell nuclei. This precipitate can be resuspended with a small amount of the third separation solution or an appropriate buffer for downstream experiments. For example, in a typical experiment, centrifuging approximately 5 ml of the final filtrate at 10,000 rpm for 12 minutes yields a white nuclear precipitate of approximately 50 to 150 μL. After staining and observation, this precipitate shows intact nuclear morphology and minimal impurities, making it suitable for direct use in subsequent studies such as DNA extraction, histone modification analysis, or chromatin immunoprecipitation. In one possible implementation, for downstream applications requiring higher purity, a third washing step with a low concentration of Triton X-100 can be added before the final centrifugation to further remove any remaining Percoll particles and trace amounts of membrane debris, resulting in a nearly pure nuclear preparation under an optical microscope.

[0039] To further verify the technical effects that the present invention can produce, the following experiments were conducted: Take 0.1g of dehydrated mature cotton boll tissue (including the seed coat) and grind it into a fine powder in a mortar under liquid nitrogen protection.

[0040] Add the finely powdered tissue to a pre-cooled centrifuge tube containing 3 mL of the first separation solution, mix with a hand homogenizer for 3 seconds, and then let stand on ice for 3 minutes.

[0041] The first separation solution consisted of 10 mM Tris-HCl (pH: 8), 50 mM KCl, and 0.5 mM spermidine. The mixture was filtered using a filter with a pore size of 40 μm (as the first pore size filter), and the filtrate was collected into a new centrifuge tube.

[0042] Centrifuge the filtrate at 4℃ and 600×g for 3 minutes and discard the supernatant.

[0043] Add 1 mL of the second separation solution (containing a non-ionic detergent) to the precipitate and slowly resuspend it by pipetting.

[0044] The second separation solution consists of: 10 mM Tris-HCl (pH: 8), 50 mM KCl, 0.5 mM spermidine, and 0.5% Tx-100. Centrifuge the resuspended solution at 4℃ and 600×g for 3 min and discard the supernatant.

[0045] Add 1 mL of washing buffer (as a high-salt washing buffer) to the precipitate and resuspend. Centrifuge at 4°C and 600×g for 3 min and discard the supernatant. Repeat this washing step twice.

[0046] Washing solution composition: 10 mM Tris-HCl (pH: 8), 150 mM NaCl, 0.5 mM spermidine; Add 0.5 mL of ice-cold third separation solution (containing components with high osmotic pressure) to the washed precipitate and slowly resuspend by blowing.

[0047] The third separation solution consisted of: 0.4 M sucrose, 30 mM Tris-HCl (pH=8), and 20 mM MgCl2. The resuspension was filtered through a 20 μm filter (as a second-pore size filter), and the filtrate was collected into a pre-cooled centrifuge tube. Then, 0.5 mL of the fourth separation solution and 0.5 mL of the fifth separation solution (two different concentrations of Percoll density gradient solutions) were slowly added to the bottom of the tube, and the tube was centrifuged at 4 °C and 2000 × g for 20 min.

[0048] The fourth separation solution consists of 15% by volume separation solution C and 75% by volume Percoll. The fifth separation solution consists of 100% Percoll. After centrifugation, the solution was observed to separate into three layers. Approximately 0.2 mL of the cell nucleus enrichment interface layer at the junction of the second and third layers was aspirated into a new centrifuge tube.

[0049] Add 0.5 mL of ice-cold third separation solution to the interface layer solution, mix by blowing, centrifuge at 4℃ and 500×g for 5 min, and discard the supernatant.

[0050] Add an appropriate amount of ice-cold third separation solution to the precipitate, resuspend the cell nuclei, and you will get the separated cotton boll cell nuclei, which can be used for subsequent staining or experiments.

[0051] A portion of the cell nucleus suspension was stained with DAPI, and the resulting counts could be used for downstream analyses such as ATAC library construction.

[0052] Microscopic examination results as follows Figure 2 As shown, the cell nuclei extracted from cotton bolls using the method described in this embodiment have a clean background with few impurities.

[0053] ATAC library quality control data are shown in Table 1: Table 1. Quality control data of ATAC library extracted from cotton bolls in Example 1 Figure 3 The quality control image of the cotton boll cell nucleus ATAC library showed that the ATAC library quality control showed a main peak at 200-400 bp. After 12 cycles of amplification, the cDNA concentration was 26.2 ng / μL and the total amount was 524 ng, which met the quality control requirements.

[0054] Because cotton boll tissues contain relatively few chloroplasts, this invention uses the milder Tx-100 to lyse cells. After lysis, the cells are washed with a separation solution that does not contain Tx-100, which maximizes the preservation of cell nuclei while ensuring effective lysis. Meanwhile, due to the high cotton fiber content in cotton bolls, this invention uses density gradient centrifugation to remove impurities, and the ratio of the impurity removal solution has been optimized.

[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for separating cell nuclei from dehydrated, mature cotton bolls, characterized in that, include: Take dehydrated mature cotton boll tissue and grind it into a fine powder under liquid nitrogen protection. The tissue must include the seed coat. The fine powder tissue is added to a pre-cooled first separation liquid containing specific components, and then subjected to a brief homogenization and ice bath standing treatment. The first separation liquid is filtered through a first-pore filter, and the filtrate is collected. The filtrate is centrifuged at low temperature, the supernatant is discarded, and the precipitate is resuspended in a second separation liquid containing a non-ionic detergent. The resuspended precipitate is centrifuged again at low temperature, the supernatant is discarded, and the precipitate is washed multiple times with a high-salt washing solution. The washed precipitate is resuspended in a third separation liquid containing a component with higher osmotic pressure. The resuspended solution is stacked on two pre-laid Percoll density gradient solutions of different concentrations and subjected to density gradient centrifugation. After centrifugation, the cell nucleus enrichment interface layer is aspirated. The interface layer solution is diluted with the third separation liquid and filtered again through a second-pore filter. The filtered solution is centrifuged at low temperature to collect the separated cotton boll cell nuclei.

2. The method as described in claim 1, characterized in that, The process involves taking dehydrated, mature cotton boll tissue and grinding it into a fine powder under liquid nitrogen protection. The tissue must contain the seed coat portion. This includes: controlling the weight of the selected cotton boll tissue to a very small range; ensuring that the selected tissue contains the seed coat portion during sampling; and rapidly grinding the cotton boll tissue containing the seed coat in liquid nitrogen until a uniform fine powder is formed.

3. The method as described in claim 1, characterized in that, The step of filtering the first separation liquid through a first-pore-size filter and collecting the filtrate includes: using a first filter with a pore size of 40 μm to perform a first coarse filtration on the separation liquid containing initially dispersed tissue; the first filter is used to retain most of the large cotton fibers and insufficiently broken tissue blocks; and collecting the filtrate after passing through the first filter as the starting material for subsequent processing.

4. The method as described in claim 1, characterized in that, The process of adding a second separation liquid containing a nonionic detergent to resuspend the precipitate includes: adding Triton X-100 to the second separation liquid based on the components of the first separation liquid; controlling the concentration of Triton X-100 at a low level to achieve mild pyrolysis; and mixing the precipitate and the second separation liquid thoroughly and evenly by slow blowing.

5. The method as described in claim 1, characterized in that, The step of stacking the resuspension onto two pre-laid Percoll density gradient solutions of different concentrations and performing density gradient centrifugation includes: pre-laying two solutions of different Percoll concentrations sequentially at the bottom of a centrifuge tube to form a discontinuous density gradient; the first layer of Percoll solution contains a higher proportion of Percoll components; the second layer of Percoll solution contains the highest proportion of Percoll components; carefully stacking the resuspension containing cell nuclei onto the top layer of the density gradient; and performing a relatively long period of medium speed and low temperature centrifugation to effectively separate the different density components in the gradient.

6. The method as described in claim 1, characterized in that, After centrifugation, the nuclear enrichment interface layer was aspirated and subsequent processing was performed, including: observing a distinct nuclear enrichment band at the interface of the two Percoll density layers after centrifugation; carefully aspirating the nuclear enrichment band into a new container; diluting the aspirated nuclear enrichment solution with a third separation solution; and finely filtering the diluted solution using a second filter with a smaller pore size; the second filter has a pore size of 20 μm.

7. The method as described in claim 1, characterized in that, The common components of the first separation solution, the second separation solution, and the washing solution include: all containing a Tris-HCl buffer system; all containing spermidine; the spermidine is used to stabilize the cell nuclear structure.

Images