Preparation kit and application method of pennisetum americana cell nucleus suspension

Abstract

The invention provides a preparation kit of a pennisetum americanum cell nucleus suspension, and relates to the field of molecular biology, the preparation kit comprises a lysis buffer system, a phase separation system, a density gradient purification system and a lysis and dyeing system; wherein the lysis buffer solution system comprises a 1 * NIB working solution and a 1 * TAPS resuspension buffer solution, and the pH value of the 1 * TAPS resuspension buffer solution is 7.8-8.2; the phase separation system adopts ATPS two-phase separation liquid; the density gradient purification system adopts a Percoll separation solution with the concentration of 33%-37%; and the lysis and staining system comprises a cell lysis solution and a DAPI staining solution. The kit can obtain a stable high-quality plant cell nucleus suspension, so that cell nucleus damage is small, the nucleus integrity rate is improved, cell nucleus fragments are reduced, cell nucleus adhesion is reduced, and a key technical support is provided for single-cell omics research of plants such as pennisetum americana and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a preparation kit of nuclear suspension of Pennisetum americanum and an application method thereof. BACKGROUND

[0002] As the main carrier of genetic information, the high-quality isolation of plant nuclei is an important foundation for carrying out molecular biology research. In the field of genomics research, the acquisition of intact nuclei plays a decisive role in ensuring the accuracy of DNA sequencing data, especially for species such as Pennisetum with complex genomes. The application of single-cell omics technology highly depends on the preparation of high-purity nuclear suspension, and the high-quality extraction of cell nuclei can effectively ensure the integrity of large fragment DNA, which is a prerequisite for successful scRNA-seq (single-cell RNA sequencing) and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis. It is also the basis for analyzing gene expression heterogeneity in biological processes such as leaf development. At the same time, the emerging plant nuclear CUT&Tag (Cleavage Under Targets and Tagmentation) technology also needs to rely on high-integrity nuclear extraction products. In the field of epigenetic research, whether it is ChIP (Chromatin Immunoprecipitation) or DNA methylation detection, the chromatin structure protected by intact nuclear membranes is required as research material. However, the traditional extraction method often fails to meet the strict requirements of modern high-throughput technologies for the purity, integrity and biological activity of cell nuclei.

[0003] Pennisetum, as a typical C4 photosynthesis model plant and high-quality forage resource, has unique value in multiple research fields; its high biomass characteristics, outstanding drought tolerance, and rich genetic diversity make it an ideal experimental material for stress resistance gene mining and biological evolution, and it has important application prospects in bioenergy development and forage quality improvement. The genome size of this species is about 2 Gb, and the Scaffold N50 reaches 193-287 Mb, which puts high requirements on the gentleness of the nuclear extraction process. At the same time, the polyphenol content in the leaf tissue of Pennisetum is as high as 8.7 mg / g, and polysaccharide substances (such as glucan) are easily co-precipitated with cell nuclei, resulting in generally low yield of traditional extraction methods.

[0004] At present, the nucleus extraction technology of Puccinellia distans mainly includes sucrose density gradient centrifugation, mechanical grinding + filtration method, enzymatic method, high pressure difference breaking method and commercial reagent kit. The sucrose density gradient centrifugation realizes the effective separation of cell organelles by using the density difference of sucrose solution, but due to the rich polysaccharide substances in the cells of Puccinellia distans, the viscosity of the medium is greatly increased, so that the recovery rate of the cell nucleus is reduced to less than 30%; at the same time, the sucrose density gradient centrifugation has low removal efficiency of polyphenol-protein complex (such as proanthocyanidin), which seriously affects the data quality of subsequent experiments. Due to the tough fiber cell wall of Puccinellia distans, the mechanical grinding process of the mechanical grinding + filtration method is easy to cause mechanical damage to the nuclear membrane, resulting in a nuclear integrity rate of less than 50%; in addition, the conventional 40 μm filter screen cannot effectively intercept the starch particles of 15-30 μm, resulting in serious background interference when performing flow cytometry detection, which significantly reduces the signal-to-noise ratio of the experiment. The enzymatic method can obtain cell nuclei with high purity, but the enzymatic process takes a long time (usually 24-72 h) and the cost of enzyme preparation is high, and in addition, the special cell wall composition (such as high siliconization) of Puccinellia distans requires special optimization of the enzymatic conditions, thereby greatly increasing the experimental difficulty and cost. The instantaneous high pressure of the high pressure difference breaking method can effectively lyse the cells, but the impact force generated thereby will irreversibly damage the integrity of the nuclear membrane, so it is completely unsuitable for epigenetic research which needs to maintain the natural structure of chromatin. The reagent kit can obtain stable and high-quality plant cell nuclei, and has small damage to the cell nuclei and good nuclear integrity rate, but the currently marketed reagent kit is mainly optimized for model plants such as Arabidopsis thaliana, and has low clarification efficiency for the high content of polyphenols (5-8% of dry weight) and polysaccharides in Puccinellia distans; at the same time, the currently marketed reagent kit is expensive, which significantly reduces the cost performance of the test and increases the test cost. SUMMARY

[0005] In view of the problems existing in the prior art, the purpose of the present application is to provide a preparation kit for Puccinellia distans cell nucleus suspension, which can obtain stable and high-quality plant cell nucleus suspension, has small damage to the cell nuclei, improves the nuclear integrity rate, reduces the cell nucleus fragments and reduces the cell nucleus adhesion, thereby significantly increasing the number of cell nuclei, and providing key technical support for single cell omics research of Puccinellia distans and similar plants with high polyphenols and polysaccharides.

[0006] The purpose of the present application is realized by the following technical solutions: A preparation kit of a Puccinellia distans cell nucleus suspension includes a lysis buffer system, a phase separation system, a density gradient purification system and a lysis and staining system; wherein the lysis buffer system includes 1×NIB (Nuclei Isolation Buffer) working solution (prepared on demand) and 1×TAPS resuspension buffer, the pH value of the 1×TAPS resuspension buffer is 7.8-8.2; the phase separation system adopts ATPS two-phase separation liquid (prepared on demand); the density gradient purification system adopts 33%-37% Percoll separation liquid; and the lysis and staining system includes cell lysis liquid (prepared on demand) and DAPI (4', 6 -diamidino-2-phenylindole) staining liquid.

[0007] Based on further optimization of the above scheme, the 1×NIB working solution is prepared from 4×NIB stock solution, which inherits 1 / 4 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, 0.8-1.2 mM of DTT (Dithiothreitol) and 0.8%-1.2% of BSA (bovine serum albumin); the 4×NIB stock solution includes 35-45 mM of MES-KOH (MES: 2-(N-morpholino) ethanesulfonic acid) with a pH value of 5.2-5.6, 35-45 mM of NaCl, 0.9-1.1 M of sucrose, 0.04%-0.06% of Tween-20 and 0.4%-0.6% of PVP-40 (polyvinylpyrrolidone-40).

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

[0009] Based on further optimization of the above scheme, the ATPS two-phase separation liquid 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 value 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 comprises 330 to 370 μL of Percoll (density gradient centrifugation medium, 15 to 30 nm colloidal silica particles surface 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 solution comprises 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 rest 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 Pennisetum americanum cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: 1 × NIB working solution is prepared from 4 × NIB stock solution (i.e. diluting all components of 4 × NIB stock solution by 1 / 4), and immediately adding spermine, spermidine, DTT and BSA; ATPS two-phase separation solution is prepared at the same time, and is layered at 2 to 8°C for 10 to 20 min for standby; fresh tender tissue of Pennisetum americanum is selected and pre-cooled on ice for 5 to 15 min; Step S2, tissue lysis and primary filtration: the prepared cell lysis solution is pre-cooled on ice for 5 to 15 min, the pre-cooled cell lysis solution is added to the pre-cooled fresh tender tissue, and the tissue is cut with a blade for 1.5 to 5 min until the tissue is fully broken; then the lysis solution is sequentially filtered through 80 to 120 μm and 35 to 45 μm filter screens to realize secondary filtration, and the filtrate is collected; Step S3, two-phase separation and enrichment: the filtrate is gently mixed with an equal volume of ATPS two-phase separation solution for 8 to 12 times, and is layered at 2 to 8°C for 10 to 20 min, and the upper phase (PEG-rich phase containing target cell nuclei) is sucked into a new centrifuge tube; Step S4, nucleus precipitation and washing: the upper phase liquid is centrifuged at 450 to 550 rcf and 2 to 8°C for 4 to 6 min, the precipitate is resuspended with 1 × NIB working solution, and filtered through an 8 to 12 μm filter screen to obtain a nucleus suspension; Step S5, density gradient purification: the nucleus suspension is loaded on top of a 33% to 37% Percoll separation solution, and centrifuged at 450 to 550 rcf and 2 to 8°C for 8 to 12 min, and the nucleus precipitate is collected at the bottom of the tube; Step S6, resuspension and quality detection: the nucleus precipitate is resuspended with 1 × TAPS resuspension buffer, and examined under a microscope by DAPI staining solution.

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

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

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

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

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

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

[0019] Based on the further optimization of the above scheme, after step S6, standardization and sub-packaging are further performed, specifically: a target amount of nuclear suspension is taken, centrifuged at 450-550 rcf and 2-8℃ for 4-6 min, and immediately used for downstream tests.

[0020] The following are the technical effects possessed by the present application: The present application realizes the "selective lysis" of membrane lysis force and nuclear protection balance through the lysis solution system composed of Triton X-100, Tween 20 and PVP-40, the high-osmotic environment provided by high-concentration sucrose, and the cationic polyamine (spermine, spermidine) to reinforce the nuclear membrane skeleton, which not only ensures that the tough cell wall is completely broken and the cell nucleus is effectively released, but also avoids damage to the cell nucleus during the lysis process. At the same time, the polar group of PVP-40 cooperates with the specific component ATPS two-phase separation solution to prevent polysaccharides and proteins on the surface of the cell nucleus from sticking, and to drive polysaccharides away from the cell nucleus rich phase, effectively removing the interference of polysaccharide contaminants and further improving the recovery rate of the cell nucleus. In addition, the present application mixes DTT, BSA, PVP-40 and the specific component ATPS two-phase separation solution in the 1×NIB working solution, cooperates with the acidic environment, forms a triple barrier to inhibit polyphenol oxidation, and constructs a defense system of "inhibiting enzyme activity-removing oxidants-adsorbing substrates", thereby avoiding problems such as cell nucleus aggregation and DNA degradation caused by polyphenol oxidants, and ensuring the integrity of nuclear DNA.

[0021] The present application uses the surface hydrophobicity and charge difference of cell nucleus and chloroplast / pieces in the two-phase system, first separates the cell nucleus from chloroplast, polysaccharide and other substances through affinity purification, and then uses density gradient for physical separation, so as to effectively reduce the residual amount of chloroplast, completely remove cell wall pieces, and further improve the purity of cell nucleus. Through the combination of MES buffer system in acidic condition and cationic polyamine (spermine, spermidine), the stability of chromatin is improved, and the specific 1xNIB working solution and 1xTAPS resuspension buffer are used, which not only improves the antioxidant capacity, provides a suitable ionic environment, but also effectively inhibits enzyme activity, avoids problems such as nuclear membrane rupture or chromatin aggregation into a group, and ensures the quality and activity of the nucleus.

[0022] The whole method of the present application takes no more than three hours, has short preparation time and high efficiency; and 3x10 5 ~1x10 6 high-purity nuclei can be obtained at a time, the nucleus integrity rate is >95%, and the chloroplast pollution is <3%, which can perfectly adapt to cell nucleus RNA-seq, ATAC-seq and flow cytometry, etc. At the same time, through the three-stage purification system of "chemical decontamination-physical phase separation-density purification", the present application realizes the standardization of high-polyphenol, polysaccharide plant cell nucleus preparation for the first time, and provides key technical support for plant single cell omics research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The flow chart for preparing the cell nucleus suspension of the example of the present application is shown.

[0024] Figure 2 The microscopic examination result of the cell nucleus suspension in Example 2 of the present application is shown.

[0025] Figure 3 The microscopic examination result of the cell nucleus suspension in Comparative Example 1 of the present application is shown.

[0026] Figure 4 The microscopic examination result of the cell nucleus suspension in Comparative Example 2 of the present application is shown. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments.

[0028] Example 1: A preparation kit of a Puccinellia distans nucleus suspension includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system; wherein the lysis buffer system includes 1×NIB (Nuclei Isolation Buffer) working solution (prepared as needed) and 1×TAPS resuspension buffer, the pH value of the 1×TAPS resuspension buffer is 7.8; the 1×NIB working solution is prepared from 4×NIB stock solution, which inherits 1 / 4 final concentration of all components of the 4×NIB stock solution (i.e., first dilute the 4×NIB stock solution by 1 / 4, and dilute 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 of spermine, 0.4 mM of spermidine, 0.8 mM of DTT (Dithiothreitol, dithiothreitol) and 0.8% of BSA (bovine serum albumin). The 4×NIB stock solution includes 35 mM of MES-KOH (MES: 2-(N-morpholino) ethanesulfonic acid) and the pH value of the MES-KOH is 5.2, 35 mM of NaCl, 0.9 M of sucrose, 0.04% of Tween-20 (Tween 20) and 0.4% of PVP-40 (polyvinylpyrrolidone-40), and the solution of the corresponding concentration is prepared by double distilled water.

[0029] The phase separation system uses ATPS two-phase separation liquid (prepared as needed), which includes 5% of PEG6000 (polyethylene glycol, average molecular weight 6000 Da), 5% of Dextran T500 (dextran, average molecular weight 500 kDa), 0.45 M of sucrose, 9 mM of MES-KOH and the pH value of the MES-KOH is 5.2, 4.5 mM of MgCl2 and 0.08% of β-mercaptoethanol, and the solution of the corresponding concentration is prepared by double distilled water.

[0030] The density gradient purification system uses 33% Percoll separation liquid, which includes 330 μL of Percoll (density gradient centrifugation medium, 15-30 nm colloidal silica particle surface wrapped with PVP) and 670 μL of 1×NIB working solution.

[0031] The lysis and staining system comprises a cell lysis solution (freshly prepared) and a DAPI (4', 6-diamidino-2-phenylindole) staining solution. The cell lysis solution comprises Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1x NIB working solution, wherein the content of Triton X-100 is 0.4%, and the rest is 1x NIB working solution; the amount of DAPI staining solution is 0.8 mg / mL.

[0032] A method for preparing a Pennisetum americanum cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: 1x NIB working solution is prepared from 4x NIB stock solution (i.e. dilute all components of 4x NIB stock solution by 1 / 4), and spermine, spermidine, DTT and BSA are immediately added according to the above formula; ATPS two-phase separation solution is prepared at the same time, and is placed at 4℃ for 10 min to stratify for standby; 0.4 g of fresh and tender P. americanum tissue is selected and pre-cooled on ice for 5 min; Step S2, tissue lysis and primary filtration: the prepared cell lysis solution is pre-cooled on ice for 5 min, 450 μL of pre-cooled cell lysis solution is added to the pre-cooled fresh and tender tissue, and a blade (such as a razor blade) is used to cut vigorously for 1.5 min until the tissue is fully broken; then the lysis solution is sequentially filtered through 80 μm and 35 μm filter screens (nylon filter screens can be used) to realize secondary filtration, and the filtrate is collected into a centrifuge tube and placed at 4℃ for 15 min; Step S3, two-phase separation and enrichment: the filtrate is gently mixed with an equal volume of ATPS two-phase separation solution for 8 times, and is placed at 4℃ for 10 min, and the upper phase (PEG-rich phase containing target cell nuclei) is sucked into a new centrifuge tube; Step S4, nucleus precipitation and washing: the upper phase solution is centrifuged at 450 rcf and 4℃ for 4 min (the centrifuge is set to slow rise and slow fall, and the same mode is used for subsequent centrifugal treatment), the precipitate is resuspended with 450 μL of 1x NIB working solution, and filtered through an 8 μm filter screen (nylon filter screen can be used) to obtain a nucleus suspension; Step S5, density gradient purification: the nucleus suspension is loaded on top of 33% Percoll separation solution, and centrifuged at 450 rcf and 4℃ for 8 min, and the nucleus precipitate at the bottom of the collection tube is collected; Step S6, resuspension and quality detection: the nucleus precipitate is resuspended with 450 μL of 1x TAPS resuspension buffer, and examined under a microscope after DAPI staining, wherein the amount of DAPI staining solution is 8 μL and 0.4 μL after resuspension with 1x TAPS resuspension buffer, and the DAPI staining solution and the nucleus suspension are dyed in the dark for 4 min. Microscopic examination includes nucleus integrity (i.e. complete roundness), purity (no chloroplast red fluorescence) and number.

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

[0034] Example 2: A preparation kit of a Puccinellia distans cell nucleus suspension includes a lysis buffer system, a phase separation system, a density gradient purification system, and a lysis and staining system; wherein the lysis buffer system includes a 1xNIB (Nuclei Isolation Buffer) working solution (prepared as needed) and a 1xTAPS resuspension buffer, the pH value of the 1xTAPS resuspension buffer is 8.0; the 1xNIB working solution is prepared from a 4xNIB stock solution, which inherits 1 / 4 of the final concentration of all components of the 4xNIB stock solution (i.e., first dilute the 4xNIB stock solution by 1 / 4, and dilute with double distilled water to dilute the concentration of all components of the 4xNIB stock solution by 1 / 4), including 2.5mL of the 4xNIB stock solution, 0.1mM of spermine, 0.5mM of spermidine, 1mM of DTT (Dithiothreitol), and 1% of BSA (bovine serum albumin). The 4xNIB stock solution includes 40mM of MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) with a pH value of 5.4, 40mM of NaCl, 1M of sucrose, 0.05% of Tween-20, and 0.5% of PVP-40, and is prepared into a solution with the corresponding concentration by adding double distilled water. The 1xTAPS resuspension buffer includes 50mM of TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 25mM of MgCl2, and is prepared into a solution with the corresponding concentration by adding double distilled water.

[0035] The phase separation system uses an ATPS two-phase separation solution (prepared as needed), which includes 6% of PEG6000 (polyethylene glycol, average molecular weight 6000Da), 6% of Dextran T500 (dextran, average molecular weight 500kDa), 0.5M of sucrose, 10mM of MES-KOH with a pH value of 5.4, 5mM of MgCl2, and 0.1% of β-mercaptoethanol, and is prepared into a solution with the corresponding concentration by adding double distilled water.

[0036] The density gradient purification system uses a 35% Percoll separation solution, which includes 350μL of Percoll (density gradient centrifugation medium, 15-30nm colloidal silica particles surface coated with PVP) and 650μL of 1xNIB working solution.

[0037] The lysis and staining system comprises a cell lysis solution (freshly prepared) and a DAPI (4', 6-diamidino-2-phenylindole) staining solution. The cell lysis solution comprises Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1x NIB working solution, wherein the content of Triton X-100 is 0.5%, and the rest is 1x NIB working solution; and the amount of DAPI staining solution is 1 mg / mL.

[0038] A method for preparing a Puccinellia distans cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: 1x NIB working solution is prepared from 4x NIB stock solution (i.e. diluting all components of 4x NIB stock solution by 1 / 4), and spermine, spermidine, DTT and BSA are immediately added according to the above formula; ATPS two-phase separation solution is prepared at the same time, and is placed at 5℃ for 15 min to stratify for standby; 0.5 g of fresh and tender Puccinellia distans tissue is selected and pre-cooled on ice for 10 min; Step S2, tissue lysis and primary filtration: the prepared cell lysis solution is pre-cooled on ice for 10 min, 500 μL of pre-cooled cell lysis solution is added to the pre-cooled fresh and tender tissue, and a blade (such as a razor blade) is used to cut vigorously for 3 min until the tissue is fully broken; then the lysis solution is sequentially filtered through 100 μm and 40 μm filter screens (nylon filter screens can be used) to realize secondary filtration, and the filtrate is collected into a centrifuge tube and placed at 4℃ for 15 min; Step S3, two-phase separation and enrichment: the filtrate is gently mixed with an equal volume of ATPS two-phase separation solution for 10 times, and is placed at 5℃ for 15 min, and the upper phase (PEG-rich phase containing target cell nuclei) is sucked into a new centrifuge tube; Step S4, nucleus precipitation and washing: the upper phase solution is centrifuged at 500 rcf and 5℃ for 5 min (the centrifuge is set to slow rise and slow fall, and the same mode is used for subsequent centrifugal treatment), the precipitate is resuspended with 500 μL of 1x NIB working solution, and filtered through a 10 μm filter screen (nylon filter screen can be used) to obtain a nucleus suspension; Step S5, density gradient purification: the nucleus suspension is loaded on top of 35% Percoll separation solution, and centrifuged at 500 rcf and 5℃ for 10 min, and the nucleus precipitate at the bottom of the collection tube is collected; Step S6, resuspension and quality detection: the nucleus precipitate is resuspended with 500 μL of 1x TAPS resuspension buffer, and examined under a microscope after DAPI staining, wherein the amount of DAPI staining solution is 10 μL and 0.5 μL after resuspension with 1x TAPS resuspension buffer, and the DAPI staining solution and the nucleus suspension are dyed in the dark for 5 min. Microscopic examination includes nucleus integrity (i.e. complete roundness), purity (no chloroplast red fluorescence) 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 adopts ATPS two-phase separation liquid (prepared immediately), and the ATPS two-phase separation liquid 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 value of 5.6, 5.5 mM MgCl2 and 0.12% β-mercaptoethanol, and the solution with corresponding concentrations is prepared by adding distilled water.

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

[0044] The lysis and staining system includes cell lysis liquid (prepared immediately) and DAPI (4', 6-diamidino-2-phenylindole) staining liquid. The cell lysis liquid includes Triton X-100 (polyoxyethylene (9.5) octylphenyl ether) and 1×NIB working solution, wherein the content of Triton X-100 is 0.6%, and the rest is 1×NIB working solution; the amount of DAPI staining liquid is 1.2 mg / mL.

[0045] A method for preparing a Pennisetum americanum cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: 1×NIB working solution is prepared from 4×NIB stock solution (i.e. all components of 4×NIB stock solution are diluted by 1 / 4), and spermine, spermidine, DTT and BSA are immediately added according to the above formula; ATPS two-phase separation liquid is prepared at the same time, and is layered at 6°C for 20 min for standby; 0.6 g of fresh and tender P. americanum tissue is selected and pre-cooled on ice for 15 min; Step S2, tissue lysis and primary filtration: the prepared cell lysis liquid is pre-cooled on ice for 15 min, 550 μL of pre-cooled cell lysis liquid is added to the pre-cooled fresh and tender tissue, and the tissue is cut with a blade (such as a razor blade) for 5 min until the tissue is fully broken; then the lysis liquid is sequentially filtered through 120 μm and 45 μm filter screens (nylon filter screens can be used), secondary filtration is realized, the filtrate is collected into a centrifuge tube, and is placed at 4°C for 15 min; Step S3, two-phase separation enrichment: the filtrate is gently mixed with an equal volume of ATPS two-phase separation liquid for 12 times, and is placed at 6°C for 20 min, and the upper phase (PEG-rich phase containing target cell nuclei) is sucked into a new centrifuge tube; Step S4, nuclear precipitation and washing: centrifuge the upper phase liquid at 550 rcf and 6°C for 6 min (the centrifuge is set to slow up and slow down, and the same method is used in subsequent centrifugal treatment), resuspend the precipitate with 1×NIB working solution, 550 μL, and filter through a 12 μm filter screen (nylon filter screen can be used) to obtain a nuclear suspension; Step S5, density gradient purification: load the nuclear suspension onto the top of 37% Percoll separation solution, centrifuge at 550 rcf and 6°C for 12 min, and collect the nuclear precipitate at the bottom of the tube; Step S6, resuspension and quality detection: resuspend the nuclear precipitate with 1×TAPS resuspension buffer, 550 μL, and perform microscopic examination with DAPI staining solution, wherein the amount of 1×TAPS resuspension buffer and DAPI staining solution after resuspension is 12 μL and 0.6 μL, respectively, and the nuclear suspension is stained with DAPI staining solution in the dark for 6 min. Microscopic examination includes nuclear integrity (i.e. complete roundness), purity (no chloroplast red fluorescence) and number.

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

[0047] Example 4: As another preferred embodiment of the present application, on the basis of any one of Examples 1 to 3, after counting after microscopic examination in step S6, standardized aliquoting is further performed, specifically: take a target amount of nuclear suspension, centrifuge at 450-550 rcf (preferably 50 rcf) and 2-8°C (preferably 5°C) for 4-6 min (preferably 5 min) to obtain a nuclear suspension of the final product, which can be immediately used for downstream experiments.

[0048] Comparative Example 1: A kit for preparing a cell nucleus suspension, comprising a lysis buffer system, a density gradient purification system, and a lysis and staining system; wherein the lysis buffer system comprises 1×NIB (Nuclei Isolation Buffer) working solution (prepared immediately before use) and 1×TAPS resuspension buffer, the pH value of the 1×TAPS resuspension buffer is 8.0; the 1×NIB working solution is prepared from 4×NIB stock solution, which inherits 1 / 4 final concentration of all components of the 4×NIB stock solution (i.e., first dilute the 4×NIB stock solution by 1 / 4, and dilute with double-distilled water to dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), and comprises 2.5 mL of 4×NIB stock solution, 0.1 mM of spermine, 0.5 mM of spermidine, 1 mM of DTT (Dithiothreitol), and 1% of BSA (bovine serum albumin). The 4×NIB stock solution comprises 40 mM of MES-KOH (MES: 2-(N-morpholino)ethanesulfonic acid) with a pH value of 5.4, 40 mM of NaCl, and 1 M of sucrose, and is prepared into a solution with corresponding concentrations by supplementing with double-distilled water. The 1×TAPS resuspension buffer comprises 50 mM of TAPS-NaOH (TAPS: N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) and 25 mM of MgCl2, and is prepared into a solution with corresponding concentrations by supplementing with double-distilled water.

[0049] The density gradient purification system is consistent with that of Embodiment 2.

[0050] The lysis and staining system comprises a cell lysis solution (prepared immediately before use) and a DAPI (4',6-diamidino-2-phenylindole) staining solution. The cell lysis solution 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 rest is 1×NIB working solution; the DAPI staining solution is consistent with that of Embodiment 2.

[0051] A method for preparing a cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: prepare 1×NIB working solution from 4×NIB stock solution (i.e., dilute the concentration of all components of the 4×NIB stock solution by 1 / 4), and immediately add spermine, spermidine, DTT, and BSA according to the above formula; select 0.5 g of fresh and tender tissue of Puccinellia distans on ice for precooling for 10 min; 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 a cell nucleus suspension, comprising a lysis buffer system, a density gradient purification system, and a lysis and staining system; wherein the lysis buffer system comprises 1xNIB (Nuclei Isolation Buffer) working solution (prepared as needed) and 1xTAPS resuspension buffer, the pH value of the 1xTAPS resuspension buffer is 8.0; the 1xNIB working solution is consistent with that in Example 2. The 4xNIB stock solution is consistent with that in Example 2. The 1xTAPS resuspension buffer is consistent with that in Example 2. The density gradient purification system is consistent with that in Example 2. The lysis and staining system is consistent with that in Example 2.

[0055] A method for preparing a cell nucleus suspension, comprising: Step S1, reagent and sample pretreatment: 1xNIB working solution is prepared from 4xNIB stock solution (i.e. the concentration of all components in the 4xNIB stock solution is diluted by 1 / 4), and spermine, spermidine, DTT and BSA are immediately added according to the above formula; 0.5g of fresh and tender tissue of Pennisetum americanum is selected and pre-cooled on ice for 10min; Step S2, tissue lysis and primary filtration: consistent with step in Example 2, to obtain a tissue suspension; Step S3, nucleus precipitation and washing: the supernatant of the tissue suspension is centrifuged at 500rcf and 5℃ for 5min (the centrifuge is set to slow up and slow down, and the same mode is adopted for subsequent centrifugal treatment), the precipitate is resuspended with 500μL of 1xNIB working solution, and filtered through a 10μm filter screen (a nylon filter screen can be used) to obtain a nucleus suspension; Step S5, density gradient purification: consistent with step in Example 2; Step S6, resuspension and quality detection: consistent with step in Example 2.

[0056] In addition, the 1xNIB working solution needs to be used within 30min after preparation.

[0057] The microscopic results are shown in Figure 4 According to Figure 4 It is concluded that the nucleus dispersibility is improved compared with Comparative Example 1, but there is still a problem of clumping; the impurities are reduced, the cellulose pollution and chloroplast residues are reduced compared with Comparative Example 1, but there are still obvious residues, and the phenomenon of obvious brown solution caused by polyphenol oxidation still exists; the yield is moderate, the proportion of intact nuclei is about 60-70%, about 1x10 5 ~3x10 5 nuclei can be obtained, which meets the number requirement of flow cytometry, but fails to meet the requirement of 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 includes 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 includes cell lysis buffer and DAPI staining solution.

2. The kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 1, characterized in that: The 1×NIB working solution is prepared from the 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 spermine, 0.4–0.6 mM spermidine, 0.8–1.2 mM DTT, and 0.8%–1.2% BSA; the 4×NIB stock solution includes 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.

3. A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 1 or 2, characterized in that: The 1×TAPS resuspension buffer comprises 45–55 mM TAPS-NaOH and 22–28 mM MgCl2.

4. A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 1 or 3, characterized in that: The ATPS two-phase separation solution comprises 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.

5. A kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 3 or 4, characterized in that: The 33%–37% Percoll separation solution comprises 330–370 μL of Percoll and 630–670 μL of 1×NIB working solution.

6. The kit for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 5, characterized in that: The cell lysis buffer includes Triton X-100 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.

7. The method for preparing cell nucleus suspension using a kit for preparing *Nymphoides oleracea* cell nucleus suspension according to claim 6, characterized in that: include: Step S1, Reagent and Sample Pretreatment: Prepare 1×NIB working solution from 4×NIB stock solution and immediately add spermine, spermidine, DTT and BSA; Prepare ATPS two-phase separation solution simultaneously, 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.

8. The method for preparing a suspension of *Nymphaea repens* cell nuclei according to claim 7, characterized in that: In step S1, the amount of fresh, tender tissue used is 0.4–0.6 g; in step S2, the amount of cell lysis buffer used is 450–550 μL.

9. The method for preparing a suspension of *Nymphoides oleracea* cell nuclei according to claim 7, characterized in that: The amount of 1×NIB working solution used for resuspension in step S4 is 450–550 μL.

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

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