CN116286594A - Method for preparing cotton root single-cell nucleus suspension for single-cell nucleus sequencing - Google Patents
Method for preparing cotton root single-cell nucleus suspension for single-cell nucleus sequencing Download PDFInfo
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Abstract
The invention relates to a method for preparing cotton root single-cell nucleus suspension for single-cell nucleus sequencing, which comprises the steps of placing cotton seeds in vermiculite sandy soil paper cups for germination and growth, removing lateral roots when first true leaves of the seeds are unfolded, and taking main root parts below hypocotyls to obtain root tissues; placing root tissues in a culture dish on ice, adding a certain amount of cutting buffer solution, and cutting the root tissues for multiple times by a blade to release cell nuclei, so as to obtain crude cell nucleus suspension; the crude cell nucleus suspension in the culture dish is collected, filtered through a nylon membrane, labeled by adding fluorescent dye, separated and centrifuged, the supernatant is discarded, and the precipitate is collected and resuspended by using cell nucleus preservation solution. The method can obtain high-quality cotton root single-cell nucleus suspension, completely meets the on-machine requirement of single-cell nucleus sequencing, solves the difficulty of releasing the root cell nucleus in the true leaf stage of cotton to a certain extent, and is vital to genetic development research and acceleration of breeding process of cotton.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing cotton root single-cell nuclear suspension for single-cell nuclear sequencing.
Background
Cotton is an important economic crop worldwide, china is one of the largest major cotton producing countries in the world, and the continuously stable or growing cotton industry has important strategic significance for sustainable economic development. Cotton quality and yield continue to decrease over the past 20 years with the growing prominence of contradictions between grain and cotton, the rapid spread of pest disease, worldwide climate change, and destruction of the soil's ecological environment. Long-term production practice shows that cultivation of stress-resistant, high-yield and high-quality cotton varieties is a powerful way for guaranteeing efficient cotton supply. However, there is still a great gap between the traditional cotton breeding process and the demand of cotton production, and the mode of molecular breeding combined with genetic engineering and the like is a necessary way to improve the quality and yield of cotton.
Cotton roots are important sites for nutrient absorption and transport, and are also the most active sites for biotic and abiotic stress responses. The rapid development period of the cotton root system is from seed germination to bud emergence, the daily growth amount of the root system can reach 2cm, the growth of overground parts is slower, the daily growth amount is only 1.4-0.5cm, and the true leaf period at the time is the period with strongest stress resistance of seedlings and is also the important period of root system establishment and bud and peach growth in the future, so that research on the gene expression regulation difference among root cells in the true leaf period of the cotton and the most positive cell population and key genes responding to stress is important for improving the stress resistance of the cotton, and simultaneously, theoretical and practical basis is provided for cotton breeding, so that the breeding process is accelerated, and the demands of cotton production and development are met.
In the age of high-speed development in the life science field, single-cell technology is one of the breakthrough with subversion and milestone significance. For plants, single-cell sequencing technology needs to involve the preparation of plant protoplasts in advance, but the long-time enzymolysis process of the method in the sample preparation process can induce the expression of stress genes, and the artificially changed cell transcription mode is changed to cause transcription preference; and due to the limitation of enzymolysis time, cell types in tissues cannot be obtained effectively, so that the obtained data cannot truly reflect the transcription states of all cells in a sample, and serious distortion of results is caused.
The single-cell nucleus suspension of the plant prepared by the prior art is mainly used for tender tissue parts, the release of cell nuclei is easy, and the release of the cell nuclei is more difficult due to thickening of cell walls of cotton root tissues in a true leaf stage. The quantity and quality of the cotton root cell nuclei obtained by the prior art can not meet the on-machine requirement of sequencing. Further improvements are needed for the preparation of single cell nuclear suspensions for cotton roots.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a method for preparing a cotton root single-cell nuclear suspension for single-cell nuclear sequencing, comprising the steps of:
(1) Acquisition of root tissue of cotton
Placing cotton seeds in a vermiculite sandy soil paper cup for germination and growth, sampling when a first true leaf of the seeds is unfolded, removing lateral roots, and taking a main root part below hypocotyls to obtain root tissues;
specifically, vermiculite sandy soil paper cup is prepared from vermiculite and sandy soil according to the following ratio of 7:3, mixing the materials according to the mass ratio, adding water, uniformly stirring until the relative humidity is 55-65%, and filling the mixture into a disposable paper cup; compared with sterilized soil, the nutrient soil prepared from vermiculite and sandy soil has strong permeability, is not easy to adhere to sticky secretion of cotton root tissues, and has less root tissue damage during sampling.
(2) Releasing root tissue nuclei
Placing root tissues in a culture dish on ice, adding a certain amount of precooled cutting buffer solution, and cutting and releasing cell nuclei for multiple times by using a blade to obtain crude cell nucleus suspension;
specifically, the cell nucleus is released by placing cotton root tissue at the junction of cutting buffer solution in a culture dish and the bottom of the culture dish by using forceps, cutting the root tissue into extremely thin slices along the cross section direction by using a sharp blade, and keeping the cut slices immersed in the cutting buffer solution all the time;
specifically, after all cotton root tissues are cut, all cut slices are intensively transferred to the middle of a culture dish, a new pre-cooled and rinsed sharp blade is replaced, the slices arranged in the middle of the culture dish are rapidly cut along one direction, the cutting times are 200-300 times, and a small amount of cutting buffer solution is added during the cutting to keep the tissues fully immersed in the buffer solution;
specifically, the cleavage buffer includes: mgSO410mmoL/L, KCL 5mmoL/L, HEPES 0.5.5 mmoL/L, DTT 1mg/mL, triton X-100.25%, RNA sin1U/uL, and solvent Nuclease-free Water.
(3) Collecting, filtering and centrifugally collecting precipitate
The crude cell nucleus suspension in the culture dish is collected, filtered through a nylon membrane, labeled by adding fluorescent dye, separated and centrifuged, the supernatant is discarded, and the precipitate is collected and resuspended by using cell nucleus preservation solution.
Specifically, the collection mode of the crude cell nucleus suspension is as follows: the method comprises the steps of using a sterilization gun head with a tip-removing wide opening to absorb root tissue fragments and thick cell nucleus suspension in a culture dish in a multiple-pass mode, passing a cell sieve with the aperture of 40 mu m into a centrifuge tube obliquely inserted in ice, then adding a small amount of cutting buffer in a multiple-pass mode to wash and collect residual root tissue fragments and thick cell nucleus suspension in the culture dish, wherein the cell sieve with the aperture of 40 mu m can filter large-particle tissue fragments, and the effect of obliquely inserting the centrifuge tube into the ice is to enable filtrate to slowly flow into the bottom of the centrifuge tube, so that cell nucleus loss is reduced.
Specifically, after the crude cell nucleus suspension is collected, the crude cell nucleus suspension is sucked by a liquid-transfering device and slowly and sequentially passes through 300-mesh nylon membranes and 600-mesh nylon membranes, and the nylon membranes can filter cell fragments larger than 20 mu m in the crude cell nucleus suspension so as to ensure that the flow cytometry channel is not blocked.
Specifically, the fluorescent dye is phenylindole dye liquor, and a proper amount of cell nuclei are sorted by a flow cytometer so as to obtain single-cell nucleus suspension.
Specifically, the components of the cell nucleus preservation solution include: mgSO410mmoL/L, KCL 5mmoL/L, HEPES 0.5.5 mmoL/L, DTT mg/mL RNAsin1U/uL, and the solvent is Nuclear-free Water.
The invention at least comprises the following beneficial effects:
the invention realizes the preparation of the cotton root mononuclear suspension for the first time, fills up the relevant blank and solves the problem of difficult extraction of cotton root cell nucleuses;
the concentration of the obtained cell nucleus reaches 4000 cells/mu L, the agglomeration rate is 7 percent, the ideal upper machine concentration requirement (3200 cells/mu L) of a single cell nucleus suspension sample for single cell nucleus sequencing is completely met, and the agglomeration rate is less than 10 percent; meets the requirement of single-cell nuclear sequencing, and provides an important way for researching the gene expression regulation difference among cotton root cells and the most active cell group and key genes responding to stress;
the invention releases the cell nucleus and firstly cuts the tissue into slices, the whole cell nucleus released in the process is sunk at the bottom of the culture dish, and then the slice tissue is transferred to the center of the culture dish for unidirectional secondary cutting, which is different from the knife cutting method in the prior art, and the released cell nucleus can greatly reduce the broken cell nucleus and fragments.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows cotton root tissue cultivated by a vermiculite sandy paper cup method in an embodiment of the invention;
FIG. 2 is a low temperature table prepared in the present invention;
FIG. 3 is a schematic representation of a crude cell nucleus suspension released in an embodiment of the invention;
FIG. 4 shows a filtered suspension of nuclei collected in an embodiment of the invention;
FIG. 5 is a 20 Xfield of view of the cell nucleus count in the practice of the present invention;
FIG. 6 is a graph showing the cell nucleus counts under 20X field of view by liquid nitrogen milling in the conventional cell nucleus releasing method;
FIG. 7 is a graph showing the nuclear count at 20 Xfield of view of a homogenate-hand-held tissue homogenizer disruption in a conventional nuclear release method;
FIG. 8 is a graph showing the nuclear count under 20X field of view by the homogenate assay in a conventional nuclear release method;
FIG. 9 shows the cell nucleus counts under a 20 Xfield of view of a conventional knife cut method in a conventional cell nucleus release method.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
Example 1
The reagents or instruments used in this example are all conventional products commercially available, and the method for preparing the cotton root single-cell nuclear suspension for single-cell nuclear sequencing in this example comprises the following steps:
(1) Manufacturing a vermiculite sandy soil paper cup: sterilizing vermiculite (2-4 mm) and sun-dried sandy soil (sieving with a 200-mesh sieve) according to the following steps: 3, adding water, stirring uniformly until the relative humidity is about 60%, and filling the mixture into a disposable paper cup to obtain the vermiculite sandy soil paper cup.
(2) Seed treatment and sowing: selecting full cotton seeds obtained by removing velvet with concentrated sulfuric acid, sterilizing for 15min in an ultra-clean workbench by 0.1% HgCl, washing with sterilized water for 3 times and 5min each time, pouring warm water (25 ℃) which is used for soaking the seeds overnight, then showering to dry, uniformly burying the seeds into vermiculite sand paper cups with the depth of 0.5cm by 6-8 grains each time, slightly pressing, covering the vermiculite sand paper cups with the humidity being 1cm thicker than that of the lower layer, and covering a mulching film, and placing the vermiculite sand paper cups in a greenhouse for growth; the growth condition of the tissue culture method is constant temperature of 28 ℃,16h illumination/8 h darkness, and the relative humidity of soil is controlled to be more than 50%; taking out the mulching film after the cotton seedlings emerge, thinning each cup of seedlings, reserving 4-5 plants, and sampling when the first true leaves grow to be unfolded.
(3) Sampling: the soil humidity is kept at about 70% the day before sampling, the paper cup is firstly gently pinched during sampling, vermiculite sand in the paper cup is enabled to become soft, the paper cup shell is torn off, the vermiculite sand attached to the cotton root is gently scattered and shaken off, the lateral root is removed, and then root tissues are placed in flowing water to be washed for 5-6 min, as shown in figure 1.
(4) Preparation work before nuclear preparation: the experimental consumables such as sharp blades, petri dishes, absorbent paper, tweezers, 40 mu m cell sieves, 300-mesh and 600-mesh nylon membranes, 1mL gun heads with sharp and wide mouths removed, measuring cylinders, beakers and the like are sterilized.
(5) Preparing a cutting buffer solution: the cutting buffer (formula of the cutting buffer is shown in Table 1) was prepared by using a 50mLRNA-free centrifuge tube, and after the volume was fixed, the cutting buffer was sterilized by filtration using a 0.22 μm filter in an ultra clean bench, and the cutting buffer was prepared as it is.
Table 1 cleavage buffer formulation:
(6) Preparation of a low-temperature workbench: placing an ice box full of crushed ice in an ultra-clean workbench, and manufacturing an inclined plane with an angle of about 15 degrees, as shown in fig. 2; and put on ice cutting buffer, sterile petri dishes, sharp blades, 40 μm cell sieves, 300 mesh and 600 mesh nylon membranes pre-chilled for 6min.
(7) Release of nuclei: 1g of cleaned cotton root tissue is placed on ice for standby, 1mL of precooled cutting buffer solution on ice is added to one side of a precooled inclined culture dish, root tissue is picked up by forceps and placed in the precooled culture dish, firstly, the cotton root tissue is placed at the junction of the cutting buffer solution and the bottom of the culture dish, the tissue is cut into 50-100 mu m slices along the cross section direction of the plant tissue by a sharp blade, the cut slices are always soaked in the cutting buffer solution, after all root tissues are cut, all the cut slices are intensively transferred to the middle of the culture dish, the slices in the middle of the culture dish are quickly cut along one direction by a new precooled and rinsed sharp blade (to keep the sharpness of the blade), a small amount of cutting buffer solution can be added during the cutting time to keep the tissues completely soaked in the buffer solution, as shown in fig. 3.
(8) Collection of crude nuclear suspension: firstly, washing a cell sieve with the aperture of 40 mu m and a 50mL round bottom centrifuge tube without enzyme by using a cutting buffer precooled on ice, then installing the cell sieve with the aperture of 40 mu m on the 50mL round bottom centrifuge tube, and obliquely inserting the centrifuge tube into ice; tissue fragments and crude cell nucleus suspension in the culture dish were carefully aspirated with a tip pipette equipped with a tip-removing wide mouth, filtered into a 50mL round bottom centrifuge tube, and the dish was rinsed by successively adding 500 μl of pre-chilled cutting buffer on ice, collected and filtered into the same 50mL round bottom centrifuge tube.
(9) Filtration of the nuclear suspension: the method comprises the steps of firstly rinsing a nylon membrane with a pore diameter of 300 meshes and 600 meshes and a 50mL round bottom centrifuge tube without enzyme by using a cutting buffer solution precooled on ice, then respectively folding the nylon membrane with the pore diameters of 300 meshes and 600 meshes into a reverse taper shape, putting the reverse taper shape on a new 50mL round bottom centrifuge tube, inserting the reverse taper shape into ice, sucking crude cell nucleus suspension by using a pipette, slowly sequentially passing through the nylon membrane with the pore diameters of 300 meshes and 600 meshes, and filtering the crude cell nucleus suspension into the 50mL round bottom centrifuge tube, thus finally obtaining 4mL cell nucleus suspension (shown in figure 4).
(10) Fluorescent dyes label the nuclei: to the obtained nucleus suspension 400. Mu.L of phenylindole dye was added to label the nuclei and incubated for 10min at 4 ℃.
(11) Sorting by a flow cytometer: sorting a proper amount of cell nuclei into an enzyme-free 2mL centrifuge tube which is rinsed with a cutting buffer by a flow cytometer; during the sorting, a cell nucleus preservation solution (the formulation of the cell nucleus preservation solution is shown in Table 2) was prepared, and after the volume was determined, the cell nucleus preservation solution was subjected to filtration sterilization by a 0.22 μm filter in an ultra clean bench, and the cell nucleus preservation solution was prepared as it is.
Table 2 nuclear preservation solution formulation:
(12) And (3) centrifugal collection: the collected single cell nucleus suspension was placed in a 4 ° horizontal centrifuge, centrifuged for 5min at 400g speed with an acceleration/deceleration gear of 1, the supernatant was discarded, and the cell nuclei were resuspended with 15uL of pre-chilled cell nucleus preservation solution.
(13) And (5) microscopic observation: dripping 5uL of the resuspended cell nucleus suspension on a cell counting plate, observing the state of the cell nucleus under a common optical microscope and a fluorescence microscope, and counting; the nuclei were oval in shape under a common light microscope and had blue fluorescence under a fluorescence microscope as shown in fig. 5.
(14) The counting method comprises the following steps: the cell counting method adopted in this example is a red blood cell counting method, the cell counting plate is 25×16 cell, and the calculation formula is: cell nucleus number/mL = cell nucleus number in 80 cells +.80×400×10000×dilution; rate of agglomeration = number of agglomerations +.f (number of nuclei + number of agglomerations). The final cotton root obtained had a complete single cell nucleus concentration of 4050 cells/. Mu.L and a clumping rate of 7% (as shown in FIG. 5).
The invention also carries out liquid nitrogen grinding, a refiner crushing method, a transverse cutting method and a traditional knife cutting method to release the cell nucleuses of the cotton roots so as to prepare single cell nuclear suspension of the cotton roots;
comparative example 1
Other conditions were the same as in example 1, except that the comparative example released the nuclei of cotton root tissue by liquid nitrogen milling, including: taking the cleaned cotton root tissue, putting the cotton root tissue into a mortar containing liquid nitrogen, quickly grinding, adding the liquid nitrogen during the process to keep the tissue completely immersed in the liquid nitrogen, grinding to white powder, and adding a cutting buffer solution.
The microscopic examination showed a large number of tissue fragments, with a total number of nuclei of 1750 cells/. Mu.L, and a clumping rate of 22% (as shown in FIG. 6).
Comparative example 2
Other conditions were the same as in example 1, except that the comparative example released the nuclei of cotton root tissue by hand-held tissue refiner disruption, comprising: the washed cotton root tissue was placed in a 50mL enzyme-free centrifuge tube with pre-chilled 1mL of cutting buffer and the nuclei were released using a hand-held tissue homogenizer at different intensities and disruption times.
The strength of the microscopic examination result is grade 2, the number of complete cell nuclei is 500 cells/. Mu.L when the breaking time is 2min, and the agglomeration rate is 50% (shown in FIG. 7).
Comparative example 3
Other conditions were the same as in example 1, except that the comparative example released the nuclei of cotton root tissue by the cross-cut method, including: placing the cleaned cotton root tissue on ice for later use, adding 1mL of precooled cutting buffer solution on ice into a precooled culture dish, carefully picking up the root tissue with forceps, placing the root tissue into the precooled culture dish, and cutting the root tissue into extremely thin slices along the cross section direction in the cutting buffer solution with a sharp blade.
The microscopic examination showed that the number of intact nuclei was 275 cells/. Mu.L and the clumping rate was 32% (as shown in FIG. 8).
Comparative example 4
Other conditions were the same as in example 1, except that the present comparative example released the nuclei of cotton root tissue by conventional knife cutting, including: placing the cleaned cotton root tissue on ice for later use, adding 1mL of precooled cutting buffer solution on ice into a precooled culture dish, carefully picking up the root tissue with forceps, placing the root tissue into the precooled culture dish, and repeatedly cutting the root tissue into fine particles in the cutting buffer solution with a sharp blade.
The microscopic examination showed that the number of intact nuclei was 2562 cells/. Mu.L and the agglomeration rate was 15% (as shown in FIG. 9).
The pre-cooling treatment selected by the invention is used for reducing RNA degradation in the preparation process of the cell nucleuses, the concentration of the cell nucleuses of the cotton roots obtained by other cell nucleuses release methods in the prior art cannot meet the single-cell nucleuses sequencing requirement, and the cutting method adopted in the embodiment 1 of the invention is the only method capable of obtaining the single-cell nucleuses suspension of the cotton roots with high quality concentration at present and meets the single-cell nucleuses sequencing requirement.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (10)
1. A method for preparing a suspension of single nuclei of cotton roots for single nucleus sequencing, comprising the steps of:
s1, obtaining root tissues:
placing cotton seeds in a vermiculite sandy soil paper cup for germination and growth, removing lateral roots when the first true leaves of the seeds are unfolded, and taking down main root parts below hypocotyls to obtain root tissues;
s2, releasing root tissue cell nuclei:
placing root tissues in a culture dish on ice, adding a certain amount of cutting buffer solution, and cutting the root tissues for multiple times by a blade to release cell nuclei, so as to obtain crude cell nucleus suspension;
s3, collecting, filtering, centrifugally collecting the precipitate
The crude cell nucleus suspension in the culture dish is collected, filtered through a nylon membrane, labeled by adding fluorescent dye, separated and centrifuged, the supernatant is discarded, and the precipitate is collected and resuspended by using cell nucleus preservation solution.
2. The method for preparing a cotton root single-cell nuclear suspension for single-cell nuclear sequencing according to claim 1, wherein in S1, vermiculite sandy paper cup is sterilized vermiculite and sandy soil according to 7:3, adding water, uniformly stirring until the relative humidity is 55-65%, and filling into a disposable paper cup.
3. The method for preparing the cotton root single-cell nucleus suspension for single-cell nucleus sequencing according to claim 1, wherein in the step S2, root tissues, a cutting buffer and a blade are placed on ice for 5-10 min for precooling before the release of cell nuclei.
4. The method for preparing a suspension of single cell nuclei of cotton roots for single cell nucleus sequencing of claim 1 wherein the petri dish is placed obliquely on ice at an angle of 10 to 30 °.
5. The method for preparing a cotton root single-cell nucleus suspension for single-cell nucleus sequencing according to claim 4, wherein in the step S2, the release mode of the cell nucleus is specifically as follows:
s21, adding 1mL of cutting buffer solution to one side of the culture dish, placing cotton root tissues in the culture dish by using forceps, placing at the junction of the cutting buffer solution and the bottom of the culture dish, cutting the root tissues into 50-100 mu m slices along the cross section direction by using a sharp blade, and keeping the cut slices immersed in the cutting buffer solution all the time;
s22, after all cotton root tissues are cut, transferring all cut slices to the middle of a culture dish in a concentrated mode, replacing a new pre-cooled and rinsed sharp blade, rapidly cutting the slices placed in the middle of the culture dish along one direction for 200-300 times, and adding a small amount of cutting buffer solution during cutting to keep the tissues completely immersed in the buffer solution.
6. The method for preparing a cotton root single-cell nuclear suspension for single-cell nuclear sequencing according to claim 1, wherein in S2, the composition of the cleavage buffer comprises: mgSO (MgSO) 4 10mmoL/L, KCL 5mmoL/L, HEPES 0.5.5 mmoL/L, DTT mg/mL, triton X-100.25%, RNAsin1U/uL, and the solvent is Nuclear-free Water.
7. The method for preparing a suspension of single cell nuclei in cotton roots for single cell nucleus sequencing of claim 1 wherein the step of collecting the suspension of coarse cell nuclei in the dish in S3 is as follows: the root tissue fragments and the crude cell nucleus suspension in the culture dish were sucked in several portions using a sterilization gun head with a wide mouth with a tip removed, and the cell sieves with a pore size of 40 μm were passed into a centrifuge tube placed obliquely in ice.
8. The method for preparing a cotton root single-cell nuclear suspension for single-cell nuclear sequencing according to claim 1, wherein in S3, after the crude nuclear suspension is collected, the crude nuclear suspension is sucked up by a pipette and filtered into a centrifuge tube through 300 mesh and 600 mesh nylon membranes in sequence.
9. The method for preparing the cotton root single-cell nucleus suspension for single-cell nucleus sequencing according to claim 1, wherein in the step S3, the fluorescent dye is phenyl indole dye solution, and the nuclei are obtained by sorting through a flow cytometer after labeling.
10. The method for preparing a cotton root single-cell nuclear suspension for single-cell nuclear sequencing according to claim 1, wherein in S3, the components of the cell nucleus preservation solution include: mgSO410mmoL/L, KCL 5mmoL/L, HEPES 0.5.5 mmoL/L, DTT 1.1 mg/mL, RNAsin1U/uL, and the solvent is Nuclear-free Water.
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| CN114807008A (en) * | 2022-06-20 | 2022-07-29 | 安阳工学院 | Preparation method and application of tomato leaf protoplast single cell suspension |
| CN114807008B (en) * | 2022-06-20 | 2023-09-29 | 安阳工学院 | Preparation method and application of tomato leaf protoplast single-cell suspension |
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