Novel grape fruit cell nucleus extraction method for single cell sequencing

Abstract

The invention discloses a novel grape fruit cell nucleus extraction method for single cell sequencing, and belongs to the technical field of cell nucleus extraction. The invention provides a method for extracting cell nucleuses of grape fruits. The method comprises the following steps: washing grape pulp tissues, carrying out light-proof enzymolysis to obtain protoplast, splitting the protoplast to obtain cell nucleuses, carrying out quality inspection on the cell nucleuses, removing impurities and the like. According to the method, the washing step is added, so that sugar impurities in the extracting solution are effectively reduced. Compared with direct nucleus extraction, the method has the advantages that the extraction process is milder, the extraction time is short, a large number of nucleuses with normal forms, high nuclear membrane integrity and few background fragments can be obtained, the method can be used for subsequent single-cell nucleus sequencing, and the sequencing result is good.

Description

Technical Field

[0001] The invention relates to the technical field of cell nucleus extraction, and in particular to a new method for extracting grape fruit cell nuclei for single-cell sequencing. Background Art

[0002] Grapes are widely grown and consumed as an economic crop in the world. Their fruit quality, growth and development mechanisms, and responses to the environment are all affected by complex molecular regulatory networks, among which sequencing technology is a key means of analyzing biological molecular information. In-depth exploration of the characteristics of grape pulp at the molecular biology level can provide a solid theoretical basis and technical support for key application areas such as variety improvement, quality improvement, and disease prevention, which is of great significance.

[0003] Traditional sequencing technologies are mostly based on whole tissues or cell populations, and it is difficult to accurately analyze the specific molecular information of different cell types in grape pulp tissue. With the rise of single-cell sequencing technology, it has achieved remarkable results in the field of animal cell research and the technology is relatively mature, but its application in plant research still faces many challenges. Grape fruit, as an important storage organ of grapes, contains a large amount of nutrients such as sugars and secondary metabolites, which makes the separation of grape fruit cell nuclei more complicated than animal cells. It is necessary to develop an efficient method for separating cell nuclei suitable for grape pulp tissue to ensure high-quality and complete single cell nuclei, while avoiding impurities interfering with subsequent sequencing reactions. Therefore, the development of a method for extracting single-cell nuclei from grape pulp suitable for different developmental stages can fill this technical gap and promote the development of grape-related biological research. Summary of the invention

[0004] The purpose of the present invention is to provide a new method for extracting grape fruit cell nuclei for single-cell sequencing to solve the problems existing in the above-mentioned prior art. The method provided by the present invention has a short extraction time, obtains a large number of complete cell nuclei, and has less background debris, thereby overcoming the defect that the existing single-cell nucleus extraction technology is not suitable for grape fruit cell nucleus extraction.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for extracting grape fruit nuclei for single-cell sequencing, comprising the following steps:

[0007] Grape pulp is taken, added with pulp tissue washing solution, washed, the pulp tissue washing solution is removed, pulp cell enzymatic hydrolysis solution is added, enzymatic hydrolysis is performed in the dark, filtered, the filtrate is collected, the supernatant is removed by centrifugation, and the mixture is resuspended with W5 solution to obtain a protoplast solution;

[0008] The protoplast solution is centrifuged to remove the supernatant, a nuclear lysis solution is added to lyse the protoplasts, the supernatant is removed by centrifugation, the solution is resuspended with a resuspension buffer, the solution is filtered with a cell sieve, the filtrate is collected, the supernatant is removed by centrifugation, the solution is resuspended with a fluorescent dye buffer to obtain a cell nuclear resuspension solution, and quality inspection is performed using a microscope and a cell counter;

[0009] The cell nucleus resuspension is removed from the cell debris and tissue debris using a flow sorter to obtain the cell nucleus resuspension.

[0010] Preferably, the grape pulp is green grape pulp or mature grape pulp.

[0011] Preferably, when the grape pulp is green grape pulp, the ingredients of the pulp tissue washing solution include: 0.5M mannitol, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol;

[0012] When the grape pulp is mature grape pulp, the components of the pulp tissue washing solution include: 0.6M mannitol, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol.

[0013] Preferably, when the grape pulp is green grape pulp, the components of the pulp cell enzymatic hydrolyzate include: 0.5M mannitol, 15μg / mL cellulase R10, 3μg / mL macerate R10, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol;

[0014] When the grape pulp is mature grape pulp, the components of the pulp cell enzymatic hydrolyzate include: 0.6M mannitol, 103μg / mL cellulase R, 103μg / mL macerate R, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol.

[0015] Preferably, the components of the nuclear lysis solution include: Tris-HCl 20mM, EDTA 1mM, NaCl 10mM, MgCl2 3mM and RNasin 40U / μL.

[0016] Preferably, the cell sieve is a 70 μm cell sieve and a 30 μm cell sieve rinsed with PBS buffer;

[0017] The components of the PBS buffer include: NaCl 135mM, KCl 2.7mM, KH2PO4 1.8mM, Na2HPO4 10mM, BSA 400μg / mL, sodium citrate 1mM, dithiothreitol 1mM and RNasin 40U / μL.

[0018] Preferably, the fluorescent dye buffer is a PBS buffer containing propidium iodide and 4',6-diamidino-2-phenylindole;

[0019] The components of the PBS buffer include: NaCl 135mM, KCl 2.7mM, KH2PO4 1.8mM, Na2HPO4 10mM, BSA 400μg / mL, sodium citrate 1mM, dithiothreitol 1mM and RNasin 40U / μL.

[0020] The present invention also provides an application of the above method in single-cell sequencing of grape fruit cells.

[0021] The present invention also provides an application of a cell nucleus obtained according to the method in single-cell sequencing of grape fruit cells.

[0022] Preferably, the single-cell sequencing is single-cell sequencing with a Q20 greater than 95% and a Q30 greater than 90%.

[0023] The present invention discloses the following technical effects:

[0024] The present invention adopts enzymatic hydrolysis to extract protoplasts and then lyse the cell nucleus. Compared with direct nuclear extraction, the extraction process is milder and can ensure good cell nuclear morphology. Figure 2 The grape pulp cell nucleus obtained by the currently commonly used direct extraction method has low nucleus integrity and the nuclear cytoplasm is translucent, which may lead to missing or abnormal information. Figure 3 The cell nucleus obtained by the method of the present invention has normal morphology and high integrity.

[0025] Since grape pulp has a high sugar content, cutting the pulp will cause the solution to contain more sugar, resulting in a viscous solution, which affects the extraction and purity of the nucleus. The present invention adds a washing step before extracting protoplasts, which can effectively reduce impurities in the extract and sugar in the solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a schematic diagram of the process of the grape fruit extraction method of the present invention;

[0028] Figure 2 This is the result of microscopic examination of grape pulp cell nuclei obtained by direct tissue extraction method;

[0029] Figure 3 This is a microscopic examination result of staining the nuclei of grape pulp cells extracted using the extraction method provided by the present invention;

[0030] Figure 4 This is the result of microscopic examination of protoplasts in Chardonnay pulp during the green fruit stage;

[0031] Figure 5 This is the result of microscopic examination of the nuclear staining of Chardonnay pulp cells in the green fruit stage;

[0032] Figure 6 This is the peak diagram of the quality inspection of the CUT&Tag library of Chardonnay pulp cell nuclei during the green fruit period;

[0033] Figure 7 This is the result of microscopic examination of protoplasts of mature Chardonnay pulp;

[0034] Figure 8 This is the result of microscopic examination of nuclear staining of Chardonnay fruit pulp at maturity;

[0035] Fig. 9 This is the peak diagram of the quality inspection of the CUT&Tag library of the nuclei of mature Chardonnay pulp. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] RNasin in the present invention is a protein with a broad-spectrum RNase inhibitory effect. The molecular weight of RNasin is about 50KD and the isoelectric point is about 4.7. It has been published in "Hou Xiaoqing. Histopathological research and identification of miRNAs and analysis of their action mechanism in grape-Botrytis cinerea interaction [D]. Northwest A&F University, 2022.".

[0042] The operation process of the method of the present invention is as follows Figure 1 shown.

[0043] Example 1 Extraction and quality inspection of Chardonnay pulp nuclei during green fruit stage

[0044] (1) Grape pulp sampling and pretreatment

[0045] Select about 10g of well-developed fresh grapes, wipe the surface of the peel clean, and put them in a clean culture dish for use; pour an appropriate amount of pre-cooled pulp tissue washing solution into the culture dish, use a clean double-sided blade and tweezers to peel and seed, and divide the pulp into small pieces of 3mm-6mm. When cutting, the blade should be sharp and the pulp should not be torn back and forth to avoid a large number of pulp cells being damaged. The dividing process should be completed in a washing solution environment as much as possible to avoid water loss in the pulp; the cut pulp is gently washed with pre-cooled pulp washing solution until the pH is 5.8 to reduce pulp cell breakage.

[0046] The formula of pulp tissue washing solution in the green fruit stage: mannitol 0.5M, 2-morpholineethanesulfonic acid 10mM, CaCl2 65mM, dithiothreitol 1mM; use KOH to adjust the pH to 5.8 and filter with a 0.45μm filter membrane before use.

[0047] (2) Grape pulp protoplast extraction

[0048] Remove the pulp washing liquid as much as possible, slowly add the pulp cell enzymolysis solution, put it into a constant temperature shaking table to avoid light and enzymolysis for 45min (25rpm, 26°C); gently draw 10μL enzymolysis solution with a cut 20μL pipette tip for microscopic examination, and if there are more complete protoplasts enzymolysis, the enzymolysis product can be filtered with a 60-mesh nylon mesh to a clean 50mL round-bottom centrifuge tube. The 60-mesh nylon mesh and the 50mL round-bottom centrifuge tube need to be rinsed in advance with precooled W5 solution to reduce the damage to the protoplasts; centrifuge the filtered enzymolysis solution at a speed of 50×g for 5min, gently remove the supernatant with a cut 1mL pipette tip, add 10mLW5 solution and gently resuspend, centrifuge again (30×g, 5min), add 5mLW5 solution, resuspend the protoplasts, and place on ice for use. The process of cleaning the protoplasts should be as gentle as possible, and it should be noted that the resuspending should be sufficient to avoid protoplast agglomeration.

[0049] The formula of W5 solution: NaCl 9.00 g / L, CaCl2·H2O 18.38 g / L, KCl 0.37 g / L, 2-morpholineethanesulfonic acid 0.46 g / L, glucose 0.50 g / L.

[0050] The formula of the enzymatic hydrolyzate of pulp cells in the green fruit stage: mannitol 0.5M, cellulase R10 (YaKult Honsha) 15μg / mL, macerate R10 (YaKult Honsha) 3μg / mL, 2-morpholineethanesulfonic acid 10mM, CaCl2 65mM, dithiothreitol 1mM; adjust the pH to 5.8 with KOH, and filter with a 0.45μm filter membrane before use.

[0051] (3) Protoplast lysis

[0052] The protoplasts were centrifuged at 4°C and 500×g for 5 min, and the supernatant was removed to obtain the protoplast precipitate; pre-cooled nuclear lysis buffer was added to lyse the protoplasts, and the cells were incubated on ice for 5-10 min. Immediately after the centrifugation, the cells were centrifuged at 4°C and 500×g for 5 min; 5 mL of resuspension buffer was added for later use.

[0053] Nuclear lysis solution formula: Tris-HCl 20mM, EDTA 1mM, NaCl 10mM, MgCl23mM, RNasin 40U / μL, pH adjusted to 7.4;

[0054] Resuspension buffer composition: Tris-HCl 20mM, NaCl 150mM, MgCl22mM, RNasin 40U / μL, 2% BSA, adjusted to pH 7.4.

[0055] (4) Extraction of fruit pulp nuclei

[0056] Rinse the 70μm and 30μm cell sieves with PBS buffer, then filter the cell nucleus resuspension, centrifuge at 500×g for 5 minutes, and discard the supernatant; resuspend the cell nuclei with 1mL of PBS buffer containing fluorescent dyes PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole), perform quality inspection under a microscope and fluorescent cell counter, incubate on ice for 5 minutes, and examine the cell nucleus extraction under a microscope.

[0057] PBS buffer formula: NaCl 135mM, KCl 2.7mM, KH2PO41.8mM, Na2HPO410mM, BSA 400μg / mL, sodium citrate 1mM, dithiothreitol 1mM, RNasin 40U / μL, pH adjusted to 7.2.

[0058] (5) Cell nucleus quality inspection and sorting

[0059] A flow cytometer is used to further remove residual cell debris and tissue fragments to obtain a considerable number of high-quality cell nuclei for subsequent single-cell nucleus library construction.

[0060] Table 1 Parameters of flow sorter

[0061]

[0062] Extraction effect and quality inspection results:

[0063] Microscopic and quality inspection results are as follows Figure 4-Figure 6 As shown, according to the method of this embodiment, the nuclei of Chardonnay pulp in the green fruit stage were extracted, the integrity of the nuclear membrane was 62%, the concentration was 1560 / μL, the volume was 50 μL, the total number was 78,000, and the agglomeration rate was 6%.

[0064] The quality inspection data of CUT&Tag library are shown in Table 2 and Table 3:

[0065] Table 2 Quality inspection data of CUT&Tag library extracted from Chardonnay pulp during green fruit period

[0066]

[0067]

[0068] Table 3 Results of smear analysis of Chardonnay pulp extraction quality inspection at green fruit stage

[0069]

[0070] Figure 6It shows that the CUT&Tag library has passed the quality inspection, with a specific peak at 760 and a main peak at 800-2000. After 14 cycles of amplification, the cDNA concentration is 2.84ng / μl and the total amount is 45.4ng, which is qualified for quality inspection. The ratio of Q20 and Q30 can be used to evaluate the quality of sequencing results. The higher the ratio, the better the quality inspection results. Table 6 shows that the cell nuclei obtained by this method are sequenced for single cell nuclei, with a Q20 of up to 95% and a Q30 of up to 90%, indicating that the sequencing results are good.

[0071] Example 2 Extraction and quality inspection of mature Chardonnay pulp nuclei

[0072] The difference from Example 1 is that the ratio of pulp tissue washing solution to pulp cell enzymatic hydrolyzate needs to be adjusted to a certain extent.

[0073] The formula of the pulp tissue washing solution for mature period: mannitol 0.6M, 2-morpholineethanesulfonic acid 10mM, CaCl2 65mM, dithiothreitol 1mM; use KOH to adjust the pH to 5.8 and filter with 0.45μm filter membrane before use.

[0074] The formula of the enzymatic hydrolyzate of pulp cells in the green fruit stage: mannitol 0.6M, cellulase R10 (YaKult Honsha) 3μg / mL, chromatase R10 (YaKult Honsha) 3μg / mL, 2-morpholineethanesulfonic acid 10mM, CaCl2 65mM, dithiothreitol 1mM; adjust the pH to 5.8 with KOH, and filter with a 0.45μm filter membrane before use.

[0075] Microscopic and quality inspection results are as follows Figure 7-Figure 9 As shown, according to the method of this example, the nuclei of mature Chardonnay pulp were extracted, the integrity of the nuclear membrane was 63%, the concentration was 1340 / μL, the volume was 50 μL, the total number was 67,000, and the agglomeration rate was 5%.

[0076] Fig. 9 It shows that the CUT&Tag library has passed the quality inspection, with a specific peak at 760 and a main peak at 800-2000. After 14 cycles of amplification, the cDNA concentration is 7.6ng / μl and the total amount is 121.6ng, which is qualified for quality inspection. The ratio of Q20 and Q30 can be used to evaluate the quality of sequencing results. The higher the ratio, the better the quality inspection result. Table 6 shows that the cell nuclei obtained by this method are sequenced for single cell nuclei, with Q2O as high as 95% and Q30 as high as 90%, indicating that the sequencing results are good.

[0077] The quality inspection data of CUT&Tag library are shown in Table 4 and Table 5:

[0078] Table 4 Quality inspection data of CUT&Tag library extracted from mature Chardonnay pulp

[0079]

[0080]

[0081] Table 5 Results of smear analysis of quality inspection of Chardonnay pulp extracted at maturity

[0082]

[0083] Table 6 Evaluation statistics of single cell nucleus sequencing data of Chardonnay pulp at green and mature stages

[0084] Sample type Total reads Total bases GC (%) N(％) Q20(％) Q30(％) Green fruit period 2,326,258,336 697,877,500,800 44.13 0.02 97.57 93.34 Maturity 2,330,727,828 699,218,348,400 45.06 0.01 97.48 93.30

[0085] The present invention adopts enzymatic hydrolysis to extract protoplasts and then lyse the cell nucleus. Compared with direct nuclear extraction, the extraction process is milder and can ensure good cell nuclear morphology. Figure 2 The grape pulp cell nucleus obtained by the currently commonly used direct extraction method has low nucleus integrity and the nuclear cytoplasm is translucent, which may lead to missing or abnormal information. Figure 3 The cell nucleus obtained by the method of the present invention has normal morphology and high integrity.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting grape fruit nuclei for single-cell sequencing, characterized in that: The following steps are involved: Grape pulp is taken, added with pulp tissue washing solution, washed, the pulp tissue washing solution is removed, pulp cell enzymatic hydrolysis solution is added, enzymatic hydrolysis is performed in the dark, filtered, the filtrate is collected, the supernatant is removed by centrifugation, and the mixture is resuspended with W5 solution to obtain a protoplast solution; The protoplast solution is centrifuged to remove the supernatant, a nuclear lysis solution is added to lyse the protoplasts, the supernatant is removed by centrifugation, the solution is resuspended with a resuspension buffer, the solution is filtered with a cell sieve, the filtrate is collected, the supernatant is removed by centrifugation, the solution is resuspended with a fluorescent dye buffer to obtain a cell nuclear resuspension solution, and quality inspection is performed using a microscope and a cell counter; The cell nucleus resuspension is removed from the cell debris and tissue debris using a flow sorter to obtain the cell nucleus resuspension.

2. The method according to claim 1, characterized in that The grape pulp is green grape pulp or mature grape pulp.

3. The method according to claim 2, characterized in that When the grape pulp is green grape pulp, the pulp tissue washing solution comprises: 0.5M mannitol, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol; When the grape pulp is mature grape pulp, the components of the pulp tissue washing solution include: 0.6M mannitol, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol.

4. The method according to claim 2, characterized in that When the grape pulp is green grape pulp, the components of the pulp cell enzymatic hydrolyzate include: 0.5M mannitol, 15μg / mL cellulase R10, 3μg / mL macerate R10, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol; When the grape pulp is mature grape pulp, the components of the pulp cell enzymatic hydrolyzate include: 0.6M mannitol, 103μg / mL cellulase R, 103μg / mL macerate R, 10mM 2-morpholineethanesulfonic acid, 65mM CaCl2 and 1mM dithiothreitol.

5. The method according to claim 1, characterized in that The components of the nuclear lysis solution include: Tris-HCl 20mM, EDTA 1mM, NaCl 10mM, MgCl2 3mM and RNasin 40U / μL.

6. The method according to claim 1, characterized in that The cell sieves are 70 μm cell sieves and 30 μm cell sieves rinsed with PBS buffer respectively; The components of the PBS buffer include: NaCl 135mM, KCl 2.7mM, KH2PO4 1.8mM, Na2HPO4 10mM, BSA 400μg / mL, sodium citrate 1mM, dithiothreitol 1mM and RNasin 40U / μL.

7. The method according to claim 1, characterized in that The fluorescent dye buffer is a PBS buffer containing propidium iodide and 4',6-diamidino-2-phenylindole; The components of the PBS buffer include: NaCl 135mM, KCl 2.7mM, KH2PO4 1.8mM, Na2HPO4 10mM, BSA 400μg / mL, sodium citrate 1mM, dithiothreitol 1mM and RNasin 40U / μL.

8. Use of the method according to any one of claims 1 to 7 in single-cell sequencing of grape fruit cells.

9. Use of a cell nucleus obtained according to the method according to any one of claims 1 to 7 in single-cell sequencing of grape fruit cells.

10. The use according to claim 9, characterized in that The single-cell sequencing is a single-cell sequencing with a Q20 greater than 95% and a Q30 greater than 90%.

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