Extraction method and application of plant cell nucleus

Abstract

The invention discloses a plant cell nucleus extraction method and application, and belongs to the technical field of plant single cell sequencing. The method comprises the following steps: carrying out tissue immobilization on plant tissues by using precooled methanol; preparing a cell nucleus lysis solution, namely an NIB solution; cleaning and chopping the fixed tissue sample, and incubating the tissue sample in an NIB solution; filtering with a screen; centrifuging, discarding supernatant, and resuspending and precipitating the NIB solution to obtain a suspension; slowly dripping the suspension into a Percoll solution, and carrying out density gradient centrifugation to purify cell nucleuses. The invention provides a high-applicability and high-quality cell nucleus extraction method which is suitable for cryopreserved or fresh tissue samples, higher cell nucleus yield and mRNA capture rate are obtained, and the data quality of single cell sequencing is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant single-cell sequencing, and in particular relates to a method for extracting plant cell nuclei and applications thereof. Background Art

[0002] Single-cell high-throughput sequencing is a revolutionary technology in modern biological research. It can reveal the heterogeneity of single cells in complex biological systems and provide a powerful tool for understanding the diversity of cell types and the dynamic changes of cell states. In the field of plant biology, the application of this technology is crucial for a deep understanding of plant development, stress adaptation mechanisms, and intercellular communication and signaling networks. Traditional plant nucleus extraction methods mainly rely on rapid grinding of fresh tissues after quick freezing in liquid nitrogen, or mincing fresh tissues in pre-prepared nuclear lysis reagents, followed by nuclear sorting and purification by filtration and flow cytometry (FACS). However, these methods have some limitations: first, most of them rely on fresh plant tissues, which limits the applicability and flexibility of the experiment; second, the flow cytometry purification protocol has high requirements for experimental conditions, which is challenging for research institutions with limited resources. Summary of the invention

[0003] The present invention aims to provide a method for extracting plant cell nuclei and its application, and provides a highly applicable and high-quality cell nucleus extraction method suitable for frozen or fresh tissue samples, so as to obtain a higher cell nucleus yield and mRNA capture rate, and improve the data quality of single-cell sequencing.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0005] A method for extracting plant cell nuclei, the method comprising the following steps:

[0006] S1. Fix the plant tissue with pre-cooled methanol and place on ice for 5 minutes;

[0007] S2, prepare cell nuclear lysis solution: NIB solution;

[0008] S3, washing and mincing the tissue sample fixed in S1, and placing it in NIB solution for incubation on ice for 5 min to obtain a lysed plant tissue solution;

[0009] S4, using a sieve to filter the plant tissue solution lysed in S3;

[0010] S5, centrifuging the plant tissue fluid filtered in S4, discarding the supernatant, and resuspending the precipitate with NIB solution to obtain a suspension;

[0011] S6. Slowly drip the suspension obtained in S5 into the Percoll solution and perform density gradient centrifugation to purify the cell nuclei.

[0012] Preferably, the plant tissue in S1 is a frozen or fresh tissue sample.

[0013] Further preferably, the frozen tissue sample includes one of Arabidopsis leaves, Chinese cabbage leaves, bamboo, and alfalfa nodules frozen in liquid nitrogen.

[0014] Further preferably, the fresh tissue sample comprises planted Arabidopsis thaliana or rice.

[0015] Preferably, the NIB solution described in S2 comprises the following components:

[0016] Tris-HCl, EDTA, KCl, Spermine-4HCl, Spermidine-3HCl, PVP 10, TritonX-100, Protease inhibitor Cocktail, RNase inhibitor, DTT, NF-Water.

[0017] Preferably, in S3, the washing specifically comprises: discarding methanol, and adding an appropriate amount of 1×PBS solution to wash the plant tissue.

[0018] Preferably, in S3, the mincing specifically comprises: discarding the 1×PBS solution, taking out the plant tissue, placing it in a clean culture dish, and mincing the plant tissue.

[0019] Preferably, in S3, an appropriate amount of NIB solution is added during the mincing process.

[0020] Preferably, the centrifugation conditions in S5 are: 4°C, 1260g centrifugation for 5 min.

[0021] Preferably, the Percoll solution in S6 is obtained by mixing Percoll and NIB solution in a volume ratio of 4:1.

[0022] Preferably, the density gradient centrifugation in S6 is specifically as follows: horizontal centrifugation at 4°C, 650g for 20-30min, aspirating the middle layer, adding NIB solution, mixing, centrifuging at 4°C, 1260g for 5min, discarding the supernatant, resuspending the precipitate with NIB solution, centrifuging at 4°C, 600g for 5min, discarding the supernatant, resuspending the precipitate with 0.04% BSA solution, centrifuging at 4°C, 600g for 5min, discarding the supernatant, and resuspending the precipitate with 0.04% BSA solution.

[0023] Further preferably, the 0.04% BSA solution contains 1 / 200 RI.

[0024] The present invention also provides an application of the plant cell nucleus prepared by the extraction method in single-cell sequencing.

[0025] The present invention also provides a single-cell sequencing method, comprising the plant cell nucleus prepared by the extraction method.

[0026] The present invention also provides a single-cell sequencing method, comprising the following steps:

[0027] T1. Using DAPI dye to count the plant cell nuclei extracted by the extraction method according to any one of claims 1 to 6;

[0028] T2. Use a high-throughput single-cell RNA library preparation kit to construct a library;

[0029] T3. Use a high-throughput sequencing platform to perform scRNA-seq and ATAC-seq sequencing.

[0030] Preferably, in T2, a high-throughput single-cell RNA library preparation kit is used to construct a library, comprising the following steps:

[0031] (1) Single-cell RNA library construction: Use the DNBelab C series high-throughput single-cell RNA library preparation kit and follow the instructions of the kit to construct a single-cell RNA library;

[0032] (2) Droplet generation: Using MGI's C4 droplet generation device, which is based on droplet microfluidics technology, through efficient mRNA capture magnetic beads and droplet recognition microbeads, ensure that each droplet contains only a single cell;

[0033] (3) Demulsification and recovery.

[0034] Preferably, the high-throughput sequencing platform in T3 is MGISEQ-2000RS or DNBSEQ-T7RS.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] (1) The present invention discloses a method for extracting plant cell nuclei and its application. This method greatly expands the application scope of single-cell sequencing technology and can be applied to a variety of plant tissues, including liquid nitrogen frozen and fresh samples. By pre-cooling methanol to fix the tissue, the original state of plant genes is effectively maintained, ensuring the accuracy and reliability of experimental data. The extraction process is simplified, the steps are concise and easy to operate, which greatly reduces the complexity and difficulty of the experiment.

[0037] (2) The present invention uses Percoll reagent to replace the traditional flow cytometry purification scheme, and purifies the cell nucleus by density gradient centrifugation, which not only improves the yield and purity of the cell nucleus, but also reduces the loss of the cell nucleus. The use of the DNBelab C series high-throughput single-cell RNA library preparation kit and the C4 droplet generation device designed by MGI significantly improves the magnetic bead recovery efficiency and the number of captured mRNAs, so that the constructed single-cell RNA library has a low doublet rate and excellent gene detection capabilities. In addition, this method maximizes the stability and repeatability of single-cell RNA library preparation, which is crucial for subsequent high-throughput sequencing analysis.

[0038] (3) Using the cell nuclei extracted by the present invention for single-cell sequencing, the number of cells, the UMI of each cell, and the gene expression of each cell nucleus are all increased, thereby improving the data output and the overall quality of the experimental results.

[0039] In summary, the technical solution disclosed in the present invention improves the efficiency and quality of plant cell nucleus extraction, reduces experimental costs and operational difficulties, and provides a more reliable and efficient experimental method for the field of plant single-cell sequencing.

[0040] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of scRNA-seq sequencing of the present invention;

[0042] Figure 2 This is a diagram of sequencing data of cell nuclei extracted from bamboo tissue in Example 1, wherein: Figure 2 A in the figure is the sudden drop of single cell of bamboo treated with methanol. Figure 2 B in the figure is the distribution map of magnetic beads in single cells of bamboo treated with methanol;

[0043] Figure 3 This is a diagram of sequencing data of cell nuclei extracted from cabbage tissue in Example 2, wherein: Figure 3 A in the figure is a sudden drop in the white menu cells treated with methanol. Figure 3 B in the figure is the distribution diagram of magnetic beads of white menu cells treated with methanol;

[0044] Figure 4 This is a sequencing data diagram of extracting cell nuclei of bamboo tissue in comparative example 1, wherein: Figure 4 A in the figure is the sudden drop of single cell of bamboo without methanol treatment. Figure 4 B in the figure is the magnetic bead distribution map of single cell of bamboo without methanol treatment;

[0045] Figure 5This is a sequencing data diagram of extracting cell nuclei of bamboo tissue in comparative example 2, wherein: Figure 5 A in the figure is the sudden drop of white menu cells without methanol treatment. Figure 5 B in the figure is the distribution diagram of magnetic beads of white menu cells without methanol treatment;

[0046] Figure 6 This is an electron micrograph of bamboo cell nuclei, with a scale of 50 μm;

[0047] Figure 7 This is an electron micrograph of cabbage cell nucleus, with a scale of 50 μm; DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0049] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0050] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0051] Example 1

[0052] Extraction method of bamboo nucleus:

[0053] S1. Select bamboo tissue and place it in a 15 mL centrifuge tube, add 1 mL of pre-cooled 100% methanol to fix the tissue, and place it on ice for 5 minutes;

[0054] S2. Prepare nuclear lysis buffer: NIB solution, prepare it on ice, the composition is shown in Table 1;

[0055] Table 1 NIB solution

[0056]

[0057]

[0058] S3, discard the methanol from the bamboo tissue fixed in S1, add an appropriate amount of 1×PBS solution to wash, discard the 1×PBS solution, take out the bamboo tissue and place it in a clean culture dish, chop the bamboo tissue with a new Geely blade, add an appropriate amount of NIB solution during the chopping process, transfer the chopped bamboo tissue to a 15mL centrifuge tube, put it in an appropriate amount of NIB solution and incubate it on ice for 5 minutes;

[0059] S4, use a 40um mesh to filter the bamboo tissue fluid cracked in S3;

[0060] S5, centrifuge the bamboo tissue fluid filtered in S4 at 1260 g for 5 min at 4°C, discard the supernatant, and resuspend the precipitate with 4 mL of NIB solution to obtain a suspension;

[0061] S6, the suspension obtained in S5 was slowly dripped into 80% Percoll solution, wherein the 80% Percoll solution was obtained by mixing 4mL Percoll and 1mL NIB solution by inverting and mixing, and density gradient centrifugation was performed as follows: 4°C, 650g horizontal centrifugation for 25min; the middle layer was sucked into a new 15mL centrifuge tube using a 1mL sterile flared pipette tip, and 5mL NIB solution was added, after sufficient mixing, 4°C, 1260g centrifugation for 5min; the supernatant was discarded, 1mL NIB solution was added to resuspend the precipitate, transferred to a 1.5mL centrifuge tube, 4°C, 600g centrifugation for 5min; the supernatant was discarded, 1mL 0.04% BSA (containing 1 / 200RI) was resuspended in the precipitate, 4°C, 600g centrifugation for 5min, and the supernatant was discarded; an appropriate amount of 0.04% BSA (containing 1 / 200RI) was added to resuspend the precipitate to obtain the bamboo cell nucleus.

[0062] scRNA-seq sequencing of bamboo cell nuclei:

[0063] T1, use DAPI dye to count the nuclei of bamboo cells;

[0064] T2. Use a high-throughput single-cell RNA library preparation kit to construct a library, including the following steps:

[0065] (1) Single-cell RNA library construction: Use the DNBelab C series high-throughput single-cell RNA library preparation kit and follow the instructions of the kit to construct a single-cell RNA library;

[0066] (2) Droplet generation: Using MGI's C4 droplet generation device, which is based on droplet microfluidics technology, through efficient mRNA capture magnetic beads and droplet recognition microbeads, ensure that each droplet contains only a single cell;

[0067] (3) Demulsification and recovery.

[0068] T3, scRNA-seq sequencing was performed using the MGISEQ-2000RS sequencing platform. The results are as follows Figure 2 .

[0069] The specific steps of Example 2 are the same as those of Example 1, except that the plant tissue in step S1 is the leaf tissue of Chinese cabbage. Figure 3 .

[0070] The specific steps of Comparative Example 1 are the same as those of Example 1, except that methanol-free treatment is performed in step S1. Figure 4.

[0071] The specific steps of Comparative Example 2 are the same as those of Example 1, except that the plant tissue in step S1 is the leaf tissue of Chinese cabbage and is treated without methanol. Figure 5 .

[0072] Result analysis:

[0073] Depend on Figure 2 and Figure 4 It can be seen that the number of cells, UMI per cell, and gene expression per cell nucleus obtained from the bamboo tissue treated with methanol in Example 1 increased. Figure 2 A single cell drop in bamboo treated with methanol Figure 4 A is a graph of the sudden drop in the single cell of bamboo without methanol treatment. By comparison, it can be seen that the data after methanol treatment in Example 1 is better.

[0074] Depend on Figure 3 and Figure 5 It can be seen that the number of cells, UMI per cell, and gene expression per cell nucleus obtained from the cabbage leaf tissue treated with methanol in Example 1 increased. Figure 3 A shows the drop in the number of white cells treated with methanol. Figure 5 A is a graph showing a sudden drop in the number of white blood cells without methanol treatment. By comparison, it can be seen that the data after methanol treatment in Example 2 is better.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for extracting plant cell nuclei, characterized in that: The extraction method comprises the following steps: S1. Fix the plant tissue with pre-cooled methanol and place on ice for 5 minutes; S2, prepare cell nuclear lysis solution: NIB solution; S3, washing and mincing the tissue sample fixed in S1, and placing it in NIB solution for incubation on ice for 5 min to obtain a lysed plant tissue solution; S4, using a sieve to filter the plant tissue solution lysed in S3; S5, centrifuging the plant tissue fluid filtered in S4, discarding the supernatant, and resuspending the precipitate with NIB solution to obtain a suspension; S6. Slowly drip the suspension obtained in S5 into the Percoll solution and perform density gradient centrifugation to purify the cell nuclei.

2. The method for extracting plant cell nuclei according to claim 1, characterized in that: The plant tissues described in S1 are frozen or fresh tissue samples.

3. The method for extracting plant cell nuclei according to claim 1, characterized in that: The NIB solution described in S2 includes the following components: Tris-HCl, EDTA, KCl, Spermine-4HCl, Spermidine-3HCl, PVP 10, TritonX-100, Proteaseinhibitor Cocktail, RNase inhibitor, DTT, NF-Water.

4. The method for extracting plant cell nuclei according to claim 1, characterized in that: The centrifugation conditions described in S5 are: 4°C, 1260 g for 5 min.

5. The method for extracting plant cell nuclei according to claim 1, characterized in that: The Percoll solution described in S6 is obtained by mixing Percoll and NIB solution in a volume ratio of 4:

1.

6. The method for extracting plant cell nuclei according to claim 1, characterized in that: The density gradient centrifugation described in S6 is specifically as follows: horizontal centrifugation at 4°C, 650g for 20-30min, aspirating the middle layer, adding NIB solution, mixing, centrifuging at 4°C, 1260g for 5min, discarding the supernatant, resuspending the precipitate with NIB solution, centrifuging at 4°C, 600g for 5min, discarding the supernatant, resuspending the precipitate with 0.04% BSA solution, centrifuging at 4°C, 600g for 5min, discarding the supernatant, and resuspending the precipitate with 0.04% BSA solution.

7. Use of the plant cell nucleus prepared by the extraction method according to any one of claims 1 to 6 in single-cell sequencing.

8. A single-cell sequencing method, characterized in that: The plant cell nucleus is prepared by the extraction method according to any one of claims 1 to 6.

9. A single-cell sequencing method, characterized in that: The steps include: T1. Using DAPI dye to count the plant cell nuclei extracted by the extraction method according to any one of claims 1 to 6; T2. Use a high-throughput single-cell RNA library preparation kit to construct a library; T3. Use a high-throughput sequencing platform to perform scRNA-seq and ATAC-seq sequencing.

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