A preparation kit of puccinilia selloana cell nucleus suspension and its application method

Abstract

The application provides a preparation kit for a Puccinellia distans cell nucleus suspension and relates to the field of molecular biology, and comprises a lysis buffer system, a phase separation system, a density gradient purification system and a lysis and staining system; wherein the lysis buffer system comprises 1xNIB working solution and 1xTAPS resuspension buffer, the pH value of the 1xTAPS resuspension buffer is 7.8-8.2; the phase separation system adopts ATPS two-phase separation solution; the density gradient purification system adopts 33%-37% Percoll separation solution; and the lysis and staining system comprises a cell lysis solution and a DAPI staining solution. The kit can obtain stable high-quality plant cell nucleus suspension, makes the cell nucleus damage small, improves the nucleus integrity rate, reduces the cell nucleus fragments and reduces the cell nucleus adhesion, and provides key technical support for plant single cell omics research of Puccinellia distans and the like.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a kit for preparing a suspension of American foxtail millet cell nuclei and its application method. Background Technology

[0002] Plant cell nuclei, as the primary carriers of genetic information, require high-quality isolation as a crucial foundation for molecular biology research. In genomics research, obtaining intact cell nuclei is essential for ensuring the accuracy of DNA sequencing data, especially for species with complex genomes such as *Pennisetum purpureus*. The application of single-cell omics technologies heavily relies on the preparation of high-purity nuclear suspensions. High-quality nuclear extraction effectively preserves the integrity of large DNA fragments, a prerequisite for successful scRNA-seq (single-cell RNA sequencing) and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analyses. It is also fundamental for understanding gene expression heterogeneity in biological processes such as leaf development. Furthermore, emerging plant cell nucleus CUT&Tag (Cleavage Under Targets and Tagmentation) technologies also depend on highly intact nuclear extraction products. In epigenetic research, both chromatin immunoprecipitation (ChIP) and DNA methylation detection require chromatin structures protected by intact nuclear membranes as research materials. However, traditional extraction methods often fail to meet the stringent requirements of modern high-throughput technologies for cell nuclear purity, integrity, and bioactivity.

[0003] Pennisetum, a typical C4 photosynthetic model plant and high-quality forage resource, possesses unique value in multiple research fields. Its high biomass, outstanding drought tolerance, and rich genetic diversity make it an ideal experimental material for discovering stress-resistance genes and biological evolution, and it holds significant application prospects in bioenergy development and forage quality improvement. The genome size of this species is approximately 2 Gb, with a Scaffold N50 ranging from 193 to 287 Mb, posing extremely high requirements for the gentleness of the nuclear extraction process. Furthermore, the polyphenol content in Pennisetum leaf tissue is as high as 8.7 mg / g, and polysaccharides (such as dextran) easily co-precipitate with the cell nucleus, resulting in generally low yields using traditional extraction methods.

[0004] Currently, the main techniques for extracting cell nuclei from *Phragmites australis* include: sucrose density gradient centrifugation, mechanical grinding + filtration, enzymatic digestion, high-pressure differential disruption, and commercial reagent kits. Sucrose density gradient centrifugation utilizes the density difference of sucrose solution to effectively separate organelles; however, because *Phragmites australis* cells are rich in polysaccharides, the viscosity of the medium increases significantly, causing the cell nucleus recovery rate to drop below 30%. Simultaneously, sucrose density gradient centrifugation has low efficiency in removing polyphenol-protein complexes (such as proanthocyanidins), severely affecting the data quality of subsequent experiments. Due to the tough fibrous cell walls of *Phragmites australis*, mechanical grinding + filtration easily causes mechanical damage to the nuclear membrane, resulting in a nuclear integrity rate of less than 50%. Furthermore, conventional 40μm filters are insufficient to effectively retain 15–30μm starch particles, causing severe background interference during flow cytometry detection and significantly reducing the experimental signal-to-noise ratio. While enzymatic digestion can yield highly pure cell nuclei, the process is time-consuming (typically 24–72 hours) and enzyme preparations are expensive. Furthermore, the unique cell wall composition of *Phragmites australis* (such as high silica content) requires specialized optimization of digestion conditions, significantly increasing experimental difficulty and cost. High-pressure differential lysis effectively lyses cells using instantaneous high pressure, but the resulting shock irreversibly damages nuclear membrane integrity, making it unsuitable for epigenetic studies requiring the preservation of natural chromatin structure. Kits can provide stable and high-quality selection of nuclei from plant-derived cells with minimal nuclear damage and good nuclear integrity. However, currently available kits are primarily optimized for model plants like *Arabidopsis thaliana*, exhibiting lower efficiency in clearing the high polyphenol content (5–8% dry weight) and polysaccharides in *Phragmites australis*. Additionally, the high price of these kits significantly reduces the cost-effectiveness of the experiment and increases overall cost. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a kit for preparing a suspension of plant nuclei from *Pennisetum purpureum* cells. This kit can obtain a stable, high-quality suspension of plant nuclei, resulting in minimal nuclear damage, improved nuclear integrity, reduced nuclear fragmentation, and decreased nuclear adhesion, thereby significantly increasing the number of nuclei. This provides key technical support for single-cell omics research on *Pennisetum purpureum* and similar plants high in polyphenols and polysaccharides.

[0006] The objective of this invention is achieved through the following technical solution: A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and 1×TAPS resuspension buffer, with the pH of the 1×TAPS resuspension buffer being 7.8–8.2. The phase separation system uses ATPS two-phase separation buffer (prepared fresh). The density gradient purification system uses 33%–37% Percoll separation solution. The lysis and staining system includes cell lysis buffer (prepared fresh) and DAPI (4',6-diamidinyl-2-phenylindole) staining solution.

[0007] Based on further optimization of the above scheme, the 1×NIB working solution is prepared from 4×NIB stock solution, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution, including: 2.3–2.7 mL of 4×NIB stock solution, 0.08–0.12 mM of spermine, 0.4–0.6 mM of spermidine, and 0.8–1.2 mM of DTT (dithiothreitol). The 4×NIB stock solution includes 35–45 mM MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) with a pH of 5.2–5.6, 35–45 mM NaCl, 0.9–1.1 M sucrose, 0.04%–0.06% Tween-20, and 0.4%–0.6% PVP-40 (polyvinylpyrrolidone-40).

[0008] Based on further optimization of the above scheme, the 1×TAPS resuspension buffer comprises 45-55 mM TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 22-28 mM MgCl2.

[0009] Based on further optimization of the above scheme, the ATPS two-phase separation solution includes 5%–7% PEG6000 (polyethylene glycol, average molecular weight 6000 Da), 5%–7% Dextran T500 (dextran, average molecular weight 500 kDa), 0.45–0.55 M sucrose, 9–11 mM MES-KOH with a pH of 5.2–5.6, 4.5–5.5 mM MgCl2, and 0.08%–0.12% β-mercaptoethanol.

[0010] Based on further optimization of the above scheme, the 33% to 37% Percoll separation solution includes 330 to 370 μL of Percoll (density gradient centrifugation medium, with 15 to 30 nm colloidal silica particles coated with PVP) and 630 to 670 μL of 1×NIB working solution.

[0011] Based on further optimization of the above scheme, the cell lysis buffer includes Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, wherein the content of Triton X-100 is 0.4% to 0.6%, and the remainder is 1×NIB working solution; the amount of DAPI staining solution is 0.8 to 1.2 mg / mL.

[0012] A method for preparing a suspension of Napier grass cell nuclei, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA; simultaneously prepare ATPS two-phase separation solution, and allow it to stand at 2-8℃ for 10-20 min for separation; select fresh young tissue of American foxtail grass and pre-cool it on ice for 5-15 min; Step S2, Tissue Lysis and Primary Filtration: Pre-cool the prepared cell lysis buffer on ice for 5-15 minutes. Add the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue and cut it vigorously with a blade for 1.5-5 minutes until the tissue is fully broken. Then, pass the lysis buffer through 80-120 μm and 35-45 μm filters to achieve secondary filtration and collect the filtrate. Step S3, Two-phase separation and enrichment: Gently mix the filtrate with an equal volume of ATPS two-phase separation solution 8 to 12 times, let it stand at 2 to 8°C for 10 to 20 minutes, and aspirate the upper phase (PEG-rich phase, containing target cell nuclei) into a new centrifuge tube; Step S4, Nuclear Precipitation and Washing: Centrifuge the supernatant at 450–550 rcf and 2–8 °C for 4–6 min. Resuspend the precipitate in 1×NIB working solution and filter it through an 8–12 μm filter to obtain the nuclear suspension. Step S5, density gradient purification: Load the nuclear suspension onto the top of 33%–37% Percoll separation medium, centrifuge at 450–550 rcf and 2–8℃ for 8–12 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, Resuspension and Quality Inspection: The nuclear precipitate was resuspended using 1×TAPS resuspension buffer and examined under a microscope using DAPI staining solution.

[0013] Based on further optimization of the above scheme, the amount of fresh young tissue used in step S1 is 0.4-0.6g; the amount of cell lysis buffer used in step S2 is 450-550μL.

[0014] Based on further optimization of the above scheme, the amount of 1×NIB working solution resuspended in step S4 is 450-550 μL.

[0015] Based on further optimization of the above scheme, the amount of 1×TAPS resuspension buffer used in step S6 is 450-550 μL.

[0016] Based on further optimization of the above scheme, in the microscopic examination of DAPI staining solution in step S6, the ratio of the volume of the suspension after resuspension with 1×TAPS resuspension buffer to the volume of DAPI staining solution is 8-12 μL: 0.4-0.6 μL.

[0017] Based on further optimization of the above scheme, in step S6, the microscopic examination includes nuclear integrity (i.e., complete spherical shape), purity (no red fluorescence of chloroplasts) and number.

[0018] Based on further optimization of the above scheme, the 1×NIB working solution and ATPS two-phase separation solution should be used within 30 minutes after preparation.

[0019] Based on further optimization of the above scheme, after step S6, standardized dispensing is also performed, specifically: take the target quantity of nuclear suspension, centrifuge at 450-550 rcf and 2-8℃ for 4-6 min, and use it immediately for downstream experiments.

[0020] The following are the technical effects of this invention: This invention utilizes a lysis buffer system composed of Triton X-100, Tween 20, and PVP-40, combined with a hypertonic environment provided by high-concentration sucrose and the reinforcement of the nuclear membrane skeleton by cationic polyamines (spermine and spermidine), to achieve "selective lysis" that balances membrane lysis force and nuclear protection. This ensures complete rupture of the tough cell wall and effective release of the nucleus while preventing damage to the nucleus during lysis. Simultaneously, the polar groups of PVP-40, in conjunction with the specific component ATPS two-phase separation buffer, prevent polysaccharides from adhering to proteins on the nuclear surface and drive polysaccharides away from the nuclear enrichment phase, effectively removing interference from polysaccharide contaminants and further improving the recovery rate of the nucleus. Furthermore, this invention uses a mixture of DTT, BSA, PVP-40, and the specific component ATPS two-phase separation buffer in a 1×NIB working solution, combined with an acidic environment, to form a triple barrier to inhibit polyphenol oxidation. This constructs a defense system of "inhibiting enzyme activity - removing oxidants - adsorbing substrates," thereby avoiding problems such as nuclear aggregation and DNA degradation caused by polyphenol oxides and ensuring the integrity of nuclear DNA.

[0021] This invention utilizes the combination of Percoll separation buffer and a specific component, ATPS, a two-phase separation buffer. Leveraging the surface hydrophobicity and charge difference between the nucleus and chloroplasts / fragments in a two-phase system, the nucleus is first separated from chloroplasts, polysaccharides, and other substances through affinity purification. Then, a density gradient is used for physical separation, effectively reducing chloroplast residue, completely removing cell wall debris, and further improving nuclear purity. The acidic MES buffer system, combined with cationic polyamines (spermine and spermidine), enhances chromatin stability. Combined with a specific 1×NIB working solution and 1×TAPS resuspension buffer, it not only improves antioxidant capacity and provides a suitable ionic environment but also effectively inhibits enzyme activity, preventing nuclear membrane rupture or chromatin aggregation, thus ensuring nuclear quality and activity.

[0022] The entire method of this invention takes no more than three hours, with short preparation time and high efficiency; and can yield 3x10⁻⁶ samples in a single batch. 5 ~1x10 6 This kit contains high-purity nuclei with a nuclear integrity rate >95% and chloroplast contamination <3%, making it perfectly compatible with nuclear RNA-seq, ATAC-seq, and flow cytometry. Furthermore, this kit utilizes a three-stage purification system of "chemical decontamination - physical phase separation - density purification," achieving, for the first time, the standardized preparation of plant cell nuclei rich in polyphenols and polysaccharides, providing crucial technical support for plant single-cell omics research. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the preparation of a suspension of American foxtail millet cell nuclei according to an embodiment of the present invention.

[0024] Figure 2 This is a microscopic examination result of the cell nucleus suspension in Example 2 of the present invention.

[0025] Figure 3 This is a microscopic examination result of the cell nucleus suspension in Comparative Example 1 of this invention.

[0026] Figure 4 This is a microscopic examination result of the cell nucleus suspension in Comparative Example 2 of this invention. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0028] Example 1: A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes a 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and a 1×TAPS resuspension buffer with a pH of 7.8. The 1×NIB working solution is prepared from a 4×NIB stock solution, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution (i.e., first diluted with 4×NIB stock solution, then diluted with double-distilled water to dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), including: 2.3 mL of 4×NIB stock solution, 0.08 mM spermine, 0.4 mM spermidine, 0.8 mM DTT (dithiothreitol), and 0.8% BSA (bovine serum albumin). The 4×NIB stock solution comprises 35 mM MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) at pH 5.2, 35 mM NaCl, 0.9 M sucrose, 0.04% Tween-20, and 0.4% PVP-40 (polyvinylpyrrolidone-40), to be made up with double-distilled water to prepare a solution of the corresponding concentration. The 1×TAPS resuspension buffer comprises 45 mM TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 22 mM MgCl2, to be made up with double-distilled water to prepare a solution of the corresponding concentration.

[0029] The phase separation system uses ATPS two-phase separation solution (prepared fresh for use). The ATPS two-phase separation solution includes 5% PEG6000 (polyethylene glycol, average molecular weight 6000 Da), 5% Dextran T500 (dextran, average molecular weight 500 kDa), 0.45 M sucrose, 9 m M MES-KOH with a pH of 5.2, 4.5 m M MgCl2, and 0.08% β-mercaptoethanol, which are replenished with double-distilled water to prepare solutions of the corresponding concentrations.

[0030] The density gradient purification system uses 33% Percoll separation medium, including 330 μL of Percoll (density gradient centrifugation medium, with 15–30 nm colloidal silica particles coated with PVP) and 670 μL of 1×NIB working solution.

[0031] The lysis and staining system includes cell lysis buffer (prepared fresh for use) and DAPI (4',6-diamidinyl-2-phenylindole) staining solution. The cell lysis buffer consists of Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, with Triton X-100 comprising 0.4% and the remainder being 1×NIB working solution; the DAPI staining solution is used at a concentration of 0.8 mg / mL.

[0032] A method for preparing a suspension of Napier grass cell nuclei, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA according to the above formula; simultaneously prepare ATPS two-phase separation solution, and let it stand at 4℃ for 10 min for separation; select 0.4 g of fresh young tissue of American foxtail grass and pre-cool it on ice for 5 min; Step S2, Tissue Lysis and Primary Filtration: Pre-cool the prepared cell lysis buffer on ice for 5 min. Add 450 μL of the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue and vigorously cut it with a blade (e.g., a razor blade) for 1.5 min until the tissue is fully broken. Then, pass the lysis buffer through 80 μm and 35 μm filters (nylon filters can be used) to achieve secondary filtration. Collect the filtrate into centrifuge tubes and let it stand at 4°C for 15 min. Step S3, Two-phase separation and enrichment: Gently mix the filtrate with an equal volume of ATPS two-phase separation solution 8 times, let it stand at 4℃ for 10 min, and aspirate the upper phase (PEG-rich phase, containing target cell nuclei) into a new centrifuge tube. Step S4, Nuclear Precipitation and Washing: Centrifuge the supernatant at 450 rcf and 4℃ for 4 min (set the centrifuge to slow rise and slow fall, and use the same method for subsequent centrifugation). Resuspend the precipitate in 450 μL of 1×NIB working solution and filter it through an 8 μm filter (a nylon filter can be used) to obtain the nuclear suspension. Step S5, density gradient purification: Load the nuclear suspension onto the top of 33% Percoll separation medium, centrifuge at 450 rcf and 4℃ for 8 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, Resuspension and Quality Assurance: The nuclear precipitate was resuspended in 450 μL of 1×TAPS resuspension buffer and examined under a microscope using DAPI staining solution. The volumes of the resuspended nucleus in 1×TAPS resuspension buffer and DAPI staining solution were 8 μL and 0.4 μL, respectively. The DAPI staining solution and the nuclear suspension were stained in the dark for 4 min. Microscopic examination included nuclear integrity (i.e., complete and round shape), purity (no red fluorescence of chloroplasts), and number.

[0033] In addition, the 1×NIB working solution and the ATPS two-phase separation solution should be used within 30 minutes after preparation.

[0034] Example 2: A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes a 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and a 1×TAPS resuspension buffer with a pH of 8.0. The 1×NIB working solution is prepared from a 4×NIB stock solution, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution (i.e., first diluted with 4×NIB stock solution, then diluted with double-distilled water to dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), including: 2.5 mL of 4×NIB stock solution, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT (dithiothreitol), and 1% BSA (bovine serum albumin). The 4×NIB stock solution comprises 40 mM MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) with a pH of 5.4, 40 mM NaCl, 1 M sucrose, 0.05% Tween-20, and 0.5% PVP-40 (polyvinylpyrrolidone-40), to be made up with double-distilled water to prepare a solution of the corresponding concentration. The 1×TAPS resuspension buffer comprises 50 mM TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 25 mM MgCl2, to be made up with double-distilled water to prepare a solution of the corresponding concentration.

[0035] The phase separation system uses ATPS two-phase separation solution (prepared fresh for use). The ATPS two-phase separation solution includes 6% PEG6000 (polyethylene glycol, average molecular weight 6000 Da), 6% Dextran T500 (dextran, average molecular weight 500 kDa), 0.5 M sucrose, 10 mM MES-KOH with a pH of 5.4, 5 mM MgCl2, and 0.1% β-mercaptoethanol, which are replenished with double-distilled water to prepare solutions of the corresponding concentrations.

[0036] The density gradient purification system uses 35% Percoll separation medium, including 350 μL of Percoll (density gradient centrifugation medium, with 15–30 nm colloidal silica particles coated with PVP) and 650 μL of 1×NIB working solution.

[0037] The lysis and staining system includes cell lysis buffer (prepared fresh for use) and DAPI (4',6-diamidinyl-2-phenylindole) staining solution. The cell lysis buffer consists of Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, with Triton X-100 comprising 0.5% and the remainder being 1×NIB working solution; the DAPI staining solution is used at a concentration of 1 mg / mL.

[0038] A method for preparing a suspension of Napier grass cell nuclei, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA according to the above formula; simultaneously prepare ATPS two-phase separation solution, and let it stand at 5℃ for 15 min for separation; select 0.5 g of fresh young tissue of American foxtail grass and pre-cool it on ice for 10 min; Step S2, Tissue Lysis and Primary Filtration: Pre-cool the prepared cell lysis buffer on ice for 10 min. Add 500 μL of the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue and use a blade (e.g., a razor blade) to vigorously cut the tissue for 3 min until the tissue is fully broken. Then, pass the lysis buffer through 100 μm and 40 μm filters (nylon filters can be used) to achieve secondary filtration. Collect the filtrate into centrifuge tubes and let it stand at 4°C for 15 min. Step S3, Two-phase separation and enrichment: Gently mix the filtrate with an equal volume of ATPS two-phase separation solution 10 times, let it stand at 5°C for 15 min, and aspirate the upper phase (PEG-rich phase, containing target cell nuclei) into a new centrifuge tube. Step S4, Nuclear Precipitation and Washing: Centrifuge the supernatant at 500 rcf and 5℃ for 5 min (set the centrifuge to slow rise and fall, and use the same method for subsequent centrifugation). Resuspend the precipitate in 500 μL of 1×NIB working solution and filter it through a 10 μm filter (a nylon filter can be used) to obtain the nuclear suspension. Step S5, density gradient purification: Load the nuclear suspension onto the top of 35% Percoll separation medium, centrifuge at 500 rcf and 5℃ for 10 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, Resuspension and Quality Assurance: The nuclear precipitate was resuspended in 500 μL of 1×TAPS resuspension buffer and examined under a microscope using DAPI staining solution. The volumes of the resuspended nucleus in 1×TAPS resuspension buffer and DAPI staining solution were 10 μL and 0.5 μL, respectively. The DAPI staining solution and the nuclear suspension were stained in the dark for 5 min. Microscopic examination included nuclear integrity (i.e., complete and round shape), purity (no red fluorescence of chloroplasts), and number.

[0039] In addition, the 1×NIB working solution and the ATPS two-phase separation solution should be used within 30 minutes after preparation.

[0040] Microscopic examination results as follows Figure 2 As shown (the microscopic examination results in Examples 1 and 3 are similar to those in Example 2), according to Figure 2 Results showed that the cell nuclei exhibited excellent monodispersity, with aggregated nuclei accounting for less than 5%, and under a microscope, they appeared as uniformly dispersed, complete, round nuclei; impurities were almost eliminated, cellulose detection was negative, chloroplast residue was minimal (±), and polyphenol content reached the lowest possible level; the yield was high and efficient, increasing to approximately 7 x 10⁻⁶. 5 The nucleus has a complete nucleus ratio of >95%, fully compatible with single-cell sequencing, ATAC-seq, CUT&Tag seq, and ChIP-seq experimental requirements.

[0041] Example 3: A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes a 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and a 1×TAPS resuspension buffer with a pH of 8.2. The 1×NIB working solution is prepared from a 4×NIB stock solution, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution (i.e., first diluted with 4×NIB stock solution, then diluted with double-distilled water to dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), including: 2.7 mL of 4×NIB stock solution, 0.12 mM spermine, 0.6 mM spermidine, 1.2 mM DTT (dithiothreitol), and 1.2% BSA (bovine serum albumin). The 4×NIB stock solution comprises 45 mM MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) at pH 5.6, 45 mM NaCl, 1.1 M sucrose, 0.06% Tween-20, and 0.6% PVP-40 (polyvinylpyrrolidone-40), to be made up with double-distilled water to prepare a solution of the corresponding concentration. The 1×TAPS resuspension buffer comprises 55 mM TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 28 mM MgCl2, to be made up with double-distilled water to prepare a solution of the corresponding concentration.

[0042] The phase separation system uses ATPS two-phase separation solution (prepared fresh for use). The ATPS two-phase separation solution includes 7% PEG6000 (polyethylene glycol, average molecular weight 6000 Da), 7% Dextran T500 (dextran, average molecular weight 500 kDa), 0.55 M sucrose, 11 mM MES-KOH with a pH of 5.6, 5.5 mM MgCl2, and 0.12% β-mercaptoethanol, which are replenished with double-distilled water to prepare solutions of the corresponding concentrations.

[0043] The density gradient purification system uses 37% Percoll separation medium, including 370 μL of Percoll (density gradient centrifugation medium, with 15–30 nm colloidal silica particles coated with PVP) and 630 μL of 1×NIB working solution.

[0044] The lysis and staining system included cell lysis buffer (prepared fresh for use) and DAPI (4',6-diamidinyl-2-phenylindole) staining solution. The cell lysis buffer consisted of Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, with Triton X-100 comprising 0.6% and the remainder being 1×NIB working solution; the DAPI staining solution was used at a concentration of 1.2 mg / mL.

[0045] A method for preparing a suspension of Napier grass cell nuclei, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA according to the above formula; simultaneously prepare ATPS two-phase separation solution, and let it stand at 6℃ for 20 min for separation; select 0.6 g of fresh young tissue of American foxtail grass and pre-cool it on ice for 15 min; Step S2, Tissue Lysis and Primary Filtration: Pre-cool the prepared cell lysis buffer on ice for 15 min. Add 550 μL of the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue and vigorously cut it with a blade (e.g., a razor blade) for 5 min until the tissue is fully broken. Then, pass the lysis buffer through 120 μm and 45 μm filters (nylon filters can be used) to achieve secondary filtration. Collect the filtrate into centrifuge tubes and let it stand at 4°C for 15 min. Step S3, Two-phase separation and enrichment: Gently mix the filtrate with an equal volume of ATPS two-phase separation solution 12 times, let it stand at 6°C for 20 min, and aspirate the upper phase (PEG-rich phase, containing target cell nuclei) into a new centrifuge tube. Step S4, Nuclear Precipitation and Washing: Centrifuge the supernatant at 550 rcf and 6°C for 6 min (set the centrifuge to slow rise and fall, and use the same method for subsequent centrifugation). Resuspend the precipitate in 550 μL of 1×NIB working solution and filter it through a 12 μm filter (a nylon filter can be used) to obtain the nuclear suspension. Step S5, density gradient purification: Load the nuclear suspension onto the top of 37% Percoll separation medium, centrifuge at 550 rcf and 6℃ for 12 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, Resuspension and Quality Assurance: The nuclear precipitate was resuspended in 550 μL of 1×TAPS resuspension buffer and examined under a microscope using DAPI staining solution. The volumes of the resuspended nucleus in 1×TAPS resuspension buffer and DAPI staining solution were 12 μL and 0.6 μL, respectively. The DAPI staining solution and the nuclear suspension were stained in the dark for 6 min. Microscopic examination included nuclear integrity (i.e., complete and round shape), purity (no red fluorescence of chloroplasts), and number.

[0046] In addition, the 1×NIB working solution and the ATPS two-phase separation solution should be used within 30 minutes after preparation.

[0047] Example 4: As another preferred embodiment of the present invention, based on any one of the embodiments of Example 1 to Example 3, after counting under microscopy in step S6, standardized dispensing is also performed, specifically: take the target number of nuclear suspensions and centrifuge at 450-550 rcf (preferably 50 rcf) and 2-8°C (preferably 5°C) for 4-6 min (preferably 5 min) to obtain the final product of cell nuclear suspension, which can be used immediately for downstream experiments.

[0048] Comparative Example 1: A kit for preparing cell nuclear suspension includes a lysis buffer system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes a 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and a 1×TAPS resuspension buffer with a pH of 8.0. The 1×NIB working solution is prepared from 4×NIB stock solution, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution (i.e., first diluted with 4×NIB stock solution, then diluted with double-distilled water to dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), including: 2.5 mL of 4×NIB stock solution, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT (dithiothreitol), and 1% BSA (bovine serum albumin). The 4×NIB stock solution comprises 40 mM MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) with a pH of 5.4, 40 mM NaCl, and 1 M sucrose, to be made up with double-distilled water to prepare a solution of the corresponding concentration. The 1×TAPS resuspension buffer comprises 50 mM TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 25 mM MgCl2, to be made up with double-distilled water to prepare a solution of the corresponding concentration.

[0049] The density gradient purification system is the same as in Example 2.

[0050] The lysis and staining system includes cell lysis buffer (prepared fresh for use) and DAPI (4',6-diamidinyl-2-phenylindole) staining solution. The cell lysis buffer comprises Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, wherein the content of Triton X-100 is 0.5%, and the remainder is 1×NIB working solution; the DAPI staining solution is the same as in Example 2.

[0051] A method for preparing a cell nuclear suspension, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA according to the above formula; select 0.5g of fresh young tissue of American foxtail grass and pre-cool it on ice for 10min; Step S2, Tissue Lysis and Primary Filtration: Pre-cool the prepared cell lysis buffer on ice for 10 min. Add 500-750 μL of the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue. Vigorously cut the tissue with a blade (e.g., a razor blade) for 3 min until the tissue is fully broken and uniformly suspended. Then filter the lysis buffer through a 40 μm filter (a nylon filter can be used) to obtain a tissue suspension. Step S3, Centrifugation and Enrichment of Nuclei: Transfer the supernatant of the tissue suspension to a centrifuge tube and centrifuge at 500 rcf and 5°C for 5 min (set the centrifuge to slow rise and slow fall, and use the same method for subsequent centrifugation processes) to precipitate cell nuclei and obtain a precipitate of cell nuclei; add 500 μL of 1×NIB buffer containing 0.5% Triton X-100 to the cell nuclei precipitate to resuspend the cell nuclei, and filter them again through a 10 μm filter (a nylon filter can be used) to obtain a secondary filtrate of cell nuclei; Step S4, density gradient purification: Slowly add the secondary filtrate to the top of the container containing 35% Percoll separation solution, and centrifuge at 500 rcf and 5℃ for 10 min; Step S5, Resuspension and Quality Assay: After centrifugation, the green layer remains at the top, while the cell nuclei are at the bottom of the tube. Carefully remove all supernatant, then resuspend the cell nuclei in 500 μL of 1xTAPS buffer to obtain a nuclear suspension. Transfer the obtained nuclear suspension to a new centrifuge tube and examine it under a microscope using DAPI staining solution. The volumes of the resuspended suspension in 1×TAPS buffer and DAPI staining solution are 2 μL and 0.5 μL, respectively. Stain the DAPI staining solution and nuclear suspension in the dark for 5 min. Microscopic examination includes nuclear integrity (i.e., perfectly round), purity (no red fluorescence of chloroplasts), and number.

[0052] In addition, the 1×NIB working solution must be used within 30 minutes of preparation.

[0053] Microscopic examination results as follows Figure 3 As shown, according to Figure 3 The results showed that this method had significant defects in extraction efficiency. Cell nuclei were severely adhered, with over 50% forming irreversible clumps, and large aggregates were clearly visible under a microscope. Impurity contamination was prominent, with a large amount of cellulose fragments and chloroplasts remaining in the sample. Polyphenol oxidation caused the solution to turn a distinct brown color, and approximately 1 x 10^6 cell nuclei were obtained. 5 One, but did not meet the requirements of basic flow cytometry.

[0054] Comparative Example 2: A kit for preparing cell nuclear suspension includes a lysis buffer system, a density gradient purification system, and a lysis and staining system. The lysis buffer system includes a 1×NIB (Nuclei Isolation Buffer) working solution (prepared fresh) and a 1×TAPS resuspension buffer, the pH of which is 8.0. The 1×NIB working solution is the same as in Example 2. The 4×NIB stock solution is the same as in Example 2. The 1×TAPS resuspension buffer is the same as in Example 2. The density gradient purification system is the same as in Example 2. The lysis and staining system is the same as in Example 2.

[0055] A method for preparing a cell nuclear suspension, comprising: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute all components of 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT and BSA according to the above formula; select 0.5g of fresh young tissue of American foxtail grass and pre-cool it on ice for 10min; Step S2, Tissue lysis and primary filtration: Same as in Example 2, to obtain a tissue suspension; Step S3, Nuclear Precipitation and Washing: Centrifuge the tissue suspension supernatant at 500 rcf and 5℃ for 5 min (set the centrifuge to slow rise and fall, and use the same method for subsequent centrifugation). Resuspend the precipitate in 500 μL of 1×NIB working solution and filter it through a 10 μm filter (a nylon filter can be used) to obtain the nuclear suspension. Step S5, density gradient purification: consistent with the steps in Example 2; Step S6, Resuspension and Quality Inspection: Same as the steps in Example 2.

[0056] In addition, the 1×NIB working solution must be used within 30 minutes of preparation.

[0057] Microscopic examination results as follows Figure 4 As shown, according to Figure 4 The results showed that the nuclear dispersion was improved compared to Comparative Example 1, but clumping issues still existed; impurities were reduced, and cellulose contamination and chloroplast residue were decreased compared to Comparative Example 1, but significant residues still remained; the phenomenon of polyphenol oxidation causing a distinct brown color in the solution still existed; the yield was moderate, with the proportion of intact nuclei around 60-70%, yielding approximately 1 x 102 5 ~3x10 5 It has a number of nuclei, which meets the requirements for flow cytometry, but not for high-throughput sequencing, and cannot be used for ATAC-seq, CUT&Tag seq, and ChIP-seq experiments.

Claims

1. A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei, characterized in that: The system includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system. The lysis buffer system consists of 1×NIB working solution and 1×TAPS resuspension buffer, with the pH of the 1×TAPS resuspension buffer being 7.8–8.

2. The phase separation system uses ATPS two-phase separation buffer. The density gradient purification system uses 33%–37% Percoll separation buffer. The lysis and staining system consists of cell lysis buffer and DAPI staining solution. The 1×NIB working solution is prepared from 4×NIB stock solution with spermine, spermidine, DTT, and BSA, inheriting 1 / 4 of the final concentration of all components of the 4×NIB stock solution. The 1×NIB working solution consists of 2.3–2.7 mL of 4×NIB stock solution, 0.08–0.12 mM spermine, 0.4–0.6 mM spermidine, 0.8–1.2 mM DTT, and 0.8%–1.2% BSA; 4×NIB... The IB stock solution consists of 35–45 mM MES-KOH with a pH of 5.2–5.6, 35–45 mM NaCl, 0.9–1.1 M sucrose, 0.04%–0.06% Tween-20, and 0.4%–0.6% PVP-40; the 1×TAPS resuspension buffer consists of 45–55 mM TAPS-NaOH and 22–28 mM MgCl2. The ATPS two-phase separation buffer consists of 5%–7% PEG6000, 5%–7% Dextran T500, 0.45–0.55M sucrose, 9–11mM MES-KOH with a pH of 5.2–5.6, 4.5–5.5mM MgCl2, and 0.08%–0.12% β-mercaptoethanol. The cell lysis buffer consists of Triton X-100 and 1×NIB working solution, wherein the content of Triton X-100 is 0.4%–0.6%, and the remainder is 1×NIB working solution.

2. The kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 1, characterized in that: The 33%–37% Percoll separation solution consists of 330–370 μL of Percoll and 630–670 μL of 1×NIB working solution.

3. The kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 2, characterized in that: The amount of DAPI staining solution used is 0.8–1.2 mg / mL.

4. A method for preparing a cell nucleus suspension using the kit for preparing *Nymphoides oleracea* cell nucleus suspension according to any one of claims 1 to 3, characterized in that: include: Step S1, Reagent and Sample Pretreatment: Prepare the base solution of 1×NIB working solution from 4×NIB stock solution, and immediately add spermine, spermidine, DTT and BSA to obtain 1×NIB working solution; simultaneously prepare ATPS two-phase separation solution, and let it stand at 2-8℃ for 10-20 min for separation; select fresh young tissue of American foxtail grass and pre-cool it on ice for 5-15 min; Step S2, Tissue lysis and primary filtration: Pre-cool the prepared cell lysis buffer on ice for 5-15 minutes, add the pre-cooled cell lysis buffer to the pre-cooled fresh and tender tissue, and use a blade to cut vigorously for 1.5-5 minutes until the tissue is fully broken. The lysis solution is then passed through 80–120 μm and 35–45 μm filters in sequence to achieve secondary filtration, and the filtrate is collected. Step S3, Two-phase separation and enrichment: Gently mix the filtrate with an equal volume of ATPS two-phase separation solution 8 to 12 times, let it stand at 2 to 8°C for 10 to 20 minutes, and aspirate the upper phase into a new centrifuge tube. Step S4, Nuclear Precipitation and Washing: Centrifuge the supernatant at 450–550 rcf and 2–8 °C for 4–6 min. Resuspend the precipitate in 1×NIB working solution and filter it through an 8–12 μm filter to obtain the nuclear suspension. Step S5, density gradient purification: Load the nuclear suspension onto the top of 33%–37% Percoll separation medium, centrifuge at 450–550 rcf and 2–8℃ for 8–12 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, Resuspension and Quality Inspection: The nuclear precipitate was resuspended using 1×TAPS resuspension buffer and examined under a microscope using DAPI staining solution.

5. The method for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 4, characterized in that: In step S1, the amount of fresh, young tissue used is 0.4–0.6 g; in step S2, the amount of cell lysis buffer used is 450–550 μL.

6. The method for preparing a suspension of *Nymphaea repens* cell nuclei according to claim 4, characterized in that: The amount of 1×NIB working solution used for resuspension in step S4 is 450–550 μL.

7. The method for preparing a suspension of *Nymphaea repens* cell nuclei according to claim 4, characterized in that: In step S6, the amount of 1×TAPS resuspension buffer used is 450–550 μL.

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