CN116478917A - Animal adipose tissue cell nucleus extraction kit and preparation method and application thereof - Google Patents

Abstract

The invention discloses an animal adipose tissue cell nucleus extraction kit and a preparation method and application thereof, belonging to the technical field of molecular biology. The animal adipose tissue cell nucleus extraction kit comprises a cell nucleus lysate, wherein the lysate contains 0.3-1 mM gradient centrifugation reagent, 30-100 mM citric acid, 0.1-1 mM DTT and 0.1-1U/mu L RNase inhibitor, and is prepared by nuclease-free water; the cell nucleus purification liquid further comprises 0.2-2% BSA, 0.5-2 mM DTT and 1-3U/mu L RNase inhibitor, and the cell nucleus purification liquid is prepared by using DPBS. The kit has the advantages of easily available raw materials, low cost, good biocompatibility of reagent components, no harmful ingredients, safety and environmental protection, and is suitable for extracting adipose tissue cell nuclei. The method for extracting the cell nuclei of the adipose tissue of the animal is simple and easy to operate, and the obtained cell nuclei have complete membranes, stable gene expression, high purity and few impurities, can be used for single-cell sequencing, and has good practical application value.

Description

Animal adipose tissue cell nucleus extraction kit and preparation method and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an animal adipose tissue cell nucleus extraction kit, a preparation method and application thereof.

Background

Single cell transcriptome sequencing (scRNA-seq) is a technique that reveals all gene expression and intercellular heterogeneity within a single cell by high throughput sequencing analysis of the transcriptome at the single cell level. In recent years, single-cell RNA sequencing technology realizes analysis of heterogeneous tissue and organ gene expression patterns on single-cell level, better solves the problem of cell heterogeneity existing in traditional researches, provides a good solution for cell type identification and rare cell type discovery, maker gene screening, cell development track discussion, cell function analysis and the like, and has been widely applied to researches on different types of tissues and cell lines of various species (especially human and mouse), including normal and pathological cells and the like.

The hyperfat can cause various diseases, wherein a series of metabolic diseases such as hypertension, arteriosclerosis, obesity, genetic anemia and the like are common. At present, a mouse model is generally used for research of related diseases. With the development of single-cell transcriptome sequencing technology, it becomes possible to directly study adipocytes, but the diameter of adipocytes is larger, and the existing single-cell suspension preparation instrument has limitation on the diameter of cells and is not suitable for large-diameter cells such as adipocytes, so that the nuclear extraction research of single-cell nuclear transcription composition becomes a new outlet. Compared with common mouse tissue (such as heart, liver, kidney, lung, etc.) cell nuclei, mouse adipose tissue cell nuclei are fragile, easy to degrade, reduce RNA integrity, and easily produce impurities in the process of enucleation, so the extraction of mouse adipose tissue cell nuclei faces a great challenge.

Disclosure of Invention

In order to solve the problems of easiness in breaking, easiness in degradation, more background impurities and the like of cell nuclei in the process of cell nuclear extraction of the adipose tissue of the mice, the invention aims to provide an extraction and purification reagent and a method which can effectively extract the cell nuclei in the adipose tissue of the mice, ensure the integrity and the extraction quantity of the cell nuclei and meet the requirement of single-cell transcriptome on the machine. In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides an animal adipose tissue cell nucleus extraction kit, which comprises a cell nucleus lysate, wherein the lysate contains 0.3-1 mM gradient centrifugation reagent, 30-100 mM citric acid, 0.1-1 mM DTT and 0.1-1U/mu L RNase inhibitor, and is prepared by nuclease-free water.

In the present invention, the cell nucleus lysate refers to a component that can extract cell nuclei from tissues or cells, ensure the release of the cell nuclei from the cells, and ensure the integrity of the cell nuclei.

In some embodiments of the invention, the gradient centrifugation reagent comprises sucrose, and may further comprise at least one of polysucrose, meglumine and polyvinylpyrrolidone coated silica shell particles. In some preferred embodiments of the invention, the gradient centrifugation reagent is sucrose only. In some embodiments of the invention, the concentration of sucrose is not too great, preferably 0.5 to 0.8mM.

In some embodiments of the invention, the concentration of citric acid cannot be excessive, with a total nuclear fraction of 30% at greater than 100mM and 20% at greater than 200 mM. In some preferred embodiments of the invention, the concentration of the citric acid is 30-60 mM.

Further, the kit also comprises a cell nucleus purifying liquid, wherein the cell nucleus purifying liquid contains 0.2-2% BSA, 0.5-2 mM DTT and 1-3U/mu L RNase inhibitor, and is prepared by using DPBS.

In the present invention, a nuclear purification solution refers to a component that can keep the nuclear membrane intact during purification, and the number of nuclei is sufficient for single cell transcriptome loading.

The second aspect of the invention provides a preparation method of the animal adipose tissue cell nucleus extraction kit of the first aspect of the invention.

Optionally, adding gradient centrifugation reagent and citric acid into the nuclease-free water according to the proportion, and adding DTT and RNase inhibitor before use to obtain the cell nucleus lysate.

Optionally, adding BSA into DPBS according to the proportion, and adding DTT and RNase inhibitor before use to obtain the cell nucleus purified solution.

The third aspect of the invention provides a method for extracting the nuclei of adipose tissue of animals, comprising the following steps:

s1, obtaining 1-2 cm3 of animal adipose tissue;

s2, transferring the animal adipose tissue obtained in the step S1 into a centrifuge tube, adding 1mL of the cell nucleus lysate of claim 1, and shearing to a size of 1-2 mm 3;

s3, adding 1-3 mL of the cell nucleus lysate, and transferring to a homogenizer for grinding;

s4, adding 1-3 mL of the cell nucleus lysate, blowing, uniformly mixing, sieving, centrifuging for 3-10 min at 2-6 ℃ and 300-800 g, and removing the supernatant to obtain the animal adipose tissue cell nucleus.

Further, the method for extracting the cell nuclei of the adipose tissue of the animal further comprises the following steps:

s5, re-suspending the animal adipose tissue cell nucleus obtained in the step S4 by using the cell nucleus purification liquid of the first aspect of the invention, centrifuging at 2-6 ℃ for 3-10 min at 300-800 g, removing the supernatant, and re-suspending by using the cell nucleus purification liquid to obtain the purified animal adipose tissue cell nucleus.

In some embodiments of the invention, in step S3, during milling, 6 μl of milling solution is taken, stained with trypan blue and examined microscopically to observe the amount of nuclei released and morphology of nuclei, and milling is stopped when the cell concentration is greater than 200/μl.

In some embodiments of the invention, the animal is a mouse.

The beneficial effects of the invention are that

In view of the above, the invention provides a method for extracting and purifying single cell nuclei of adipose tissues of mice,

compared with the prior art, the invention has the following beneficial effects:

the kit has the advantages of easily available raw materials, low cost, good biocompatibility of reagent components, no harmful ingredients, safety and environmental protection, and is suitable for extracting the adipose tissue cell nuclei of mice.

The method for extracting the cell nuclei of the adipose tissue of the animal can minimize the degradation of RNA, and solves the problems of long operation time, complicated flow, easy breakage of the cell nuclei, low yield, background impurities and the like in the existing single cell nucleus extraction.

The method for extracting the cell nuclei of the adipose tissue of the animal is simple and easy to operate, and the obtained cell nuclei have complete membranes, stable gene expression, high purity and few impurities, can be used for single-cell sequencing, and has good practical application value.

Drawings

FIG. 1 shows the result of the nuclear extraction purification microscope examination in example 2 of the present invention.

FIG. 2 shows the result of fluorescence count of nuclear extraction purification in example 2 of the present invention.

FIG. 3 shows the single cell nuclear sequencing results in example 3 of the present invention.

FIG. 4 shows the result of the nuclear extraction purification microscope examination in example 4 of the present invention.

FIG. 5 shows the result of fluorescence count of nuclear extraction purification in example 4 of the present invention.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.

Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 reagent for extracting and purifying fatty tissue nuclei of mice

This example provides reagents for the nuclear extraction of mouse adipose tissue, including nuclear lysates and nuclear purification fluids.

(1) The cell nucleus lysate was formulated by adding the components shown in Table 1

TABLE 1 Nuclear lysate

And (3) uniformly mixing the components except the DTT and the RNase inhibitor, then preserving at 2-8 ℃, and adding the DTT and the RNase inhibitor before use. Wherein:

sucrose: for nuclear gradient centrifugation to remove incomplete nuclei and other cell debris;

citric acid: belongs to one of fruit acids, and can digest cell membrane gently to release cell nucleus;

DTT: is a small molecular organic reducing agent which is used as a reducing agent and a deprotection agent of sulfhydrylation DNA;

BSA: maintaining osmotic pressure, pH buffering and carrier effects, and protecting cell nucleus;

RNase inhibitor: can protect the RNA in the nucleus from degradation.

(2) The cell nucleus purified solution was formulated by adding the components shown in Table 2

TABLE 2 Nuclear purification liquid

And uniformly mixing the components except the DTT and the RNase inhibitor, then preserving at 2-8 ℃, and adding the DTT and the RNase inhibitor before use.

The cell nucleus lysate and the cell nucleus purified solution can be prepared into a kit for extracting the cell nucleus of the adipose tissue of the mouse.

Example 2 extraction and purification of mouse adipose tissue nuclei

In this example, the kit prepared in example 1 was used to extract and purify the nuclei of the adipose tissues of mice, and the specific steps are as follows:

(1) Taking 1-2 cm3 of mouse adipose tissue (fresh tissue and liquid nitrogen frozen tissue can be used);

(2) Transferring the tissue into a 5mL centrifuge tube, adding 1mL of precooled cell nucleus lysate, and shearing into fragments (1-2 mm3 in size) by using scissors;

(3) Adding 2mL of precooled cell nucleus lysate, transferring the tissue to a glass homogenizer, and grinding the tissue by using the homogenizer on ice;

(4) Standing on ice for 3min, staining with trypan blue, microscopic examination, observing the release amount of cell nucleus and the form of cell nucleus, and grinding or blowing according to tissue condition if the cell concentration is less than 100 cells/μL;

(5) Continuously adding 2mL of precooled cell nucleus lysate, blowing and mixing uniformly, sieving with a 70/30 mu m sieve, centrifuging at 4 ℃ for 5min at 500g, and collecting cell nuclei;

(6) Removing supernatant, re-suspending and cleaning with the cell nucleus purified solution, and centrifuging at 400g for 5min;

(7) The supernatant was removed, and the cells were resuspended in 1mL of nuclear purified solution and observed with a microscope and a countstar counter.

The inventors observed the morphology of the nuclei under a trypan blue staining microscope, and the results are shown in fig. 1. The inventor further uses a countstar cell counter to count AOPI fluorescence staining of the cell nucleus suspension, the result is shown in figure 2, and the total cell nucleus is 30 ten thousand, the boundary of the nuclear membrane is clear, and the whole cell nucleus accounts for 90% by combining with figures 1 and 2; the suspension background is clean and the impurities are less; the nuclei do not agglomerate, and meet the single cell uploading requirement of 10X Genomics.

EXAMPLE 3 Single cell Nuclear sequencing

The nuclear concentration prepared in example 2 was adjusted to 1000 nuclei per mu.L using the nuclear purification solution prepared in example 1. Samples were subjected to 10 x Genomics single cell transcriptome sequencing according to 8000 capture targets. As shown in FIG. 3 and Table 3, it was found from FIG. 3 and Table 3 that the single cell sequencing result was high in quality, 9206 nuclei (Estimated Number of Cells) were captured, the median value (Median Genes per Cell) of the gene was 1446, and the ratio of high quality cell reads (Fraction Reads in Cells) was 89.3%.

TABLE 3 Single cell sequencing results

Example 4 different Nuclear extraction reagents and methods for extracting the nuclei of mouse fat

To verify the superiority of the nuclear extraction reagent and method of the present invention, the inventors extracted the adipose tissue nuclei of mice using the commercially available Sigma kit (NUC 201).

Lysates and purifications from Sigma kit

Lysate configurations are shown in table 4:

TABLE 4 sigma cell nucleus lysate

Purifying liquid: nuclei PURE Storage Buffer.

The cell nucleus lysate and the purification liquid provided by the Sigma kit are used for extracting and purifying the cell nucleus of the adipose tissue of the mouse, and the specific steps are as follows:

(1) Extracting cell nuclei: taking 1g of prepared mouse adipose tissue, cleaning the adipose tissue by using DPBS, transferring the tissue into a 5mL centrifuge tube, and cutting the tissue into pieces (1-2 mm) by using scissors ³ Size), adding 3mL of precooled cell nucleus lysate, transferring the tissue to a glass homogenizer, and grinding the tissue by using the homogenizer; standing on ice for 5min, staining with trypan blue, microscopic examination, observing the release amount of cell nucleus and the form of cell nucleus, and grinding or blowing according to tissue condition if the release amount is small;

(2) And (3) cell nucleus filtration and purification: adding 3mL Nuclei PURE Storage Buffer, blowing, mixing, sieving with 70/30um sieve, centrifuging at 4deg.C for 5min at 500g, and collecting cell nuclei; removing the supernatant, lightly blowing and beating the resuspended cells by using 1mL Nuclei PURE Storage Buffer, then continuously adding 4mL Nuclei PURE Storage Buffer, lightly blowing and beating, uniformly mixing, and centrifuging for 400g and 5min;

(3) And (3) counting and observing: cells were resuspended by addition of 200ulNuclei PURE Storage Buffer, and observed with a microscope and countstar counter.

The purified nuclei were subjected to microscopic examination and AOPI staining fluorescence counting by a countstar counter, and the results are shown in fig. 4 and 5, respectively. The results show that the reagent and the method provided by the sigma kit (NUC 201) are used for extracting the adipose tissue nuclei of the mice, the effect is quite unsatisfactory, the number of broken fragments of the nuclei is large, the agglomeration is serious, the amount of the nuclei is less, the nuclei is only about 5 ten thousand, and the requirement of the single cell transcriptome on the machine is not met.

Example 5 verification of the Performance of the kit of example 1

To further verify the performance of the kit of example 1, the inventors adjusted other components or ratios, performed nuclear extraction of mouse adipose tissue according to the method of example 2, and the protocols and results are shown in table 5.

TABLE 5 effects of protocol modulation on nuclear extraction

As can be seen from table 5:

after sucrose in the cell nucleus lysate is replaced by the meglumine, the number of the extracted cell nuclei is reduced, and the whole cell nuclei accounts for 25%; further studies have found that if sucrose is replaced with polysucrose (Ficoll ^® ) Or polyvinylpyrrolidone coated silica shell particles (Percoll ^® ) The number and integrity of the extracted nuclei will also be affected, but if mixed with sucrose, the number of extracted nuclei will increase significantly and the fraction of intact nuclei will also increase significantly.

The NP40 is used for replacing citric acid in the cell nucleus lysate, the number of the extracted cell nuclei is reduced, and the whole cell nuclei account for 40 percent;

regulating the concentration of sucrose in the cell nucleus lysate to 2mM, and reducing the number of the extracted cell nuclei, wherein the whole cell nuclei account for 40%;

the concentration of citric acid in the cell nucleus lysate was adjusted to 100mM, the number of extracted cell nuclei was reduced, the whole cell nuclei was 30%, the concentration of citric acid in the cell nucleus lysate was adjusted to 200mM, the number of extracted cell nuclei was approximately equivalent, and the whole cell nuclei was 20%. Further researches show that the optimal concentration of the citric acid is 30-60 mM;

the BSA in the cell nucleus purifying liquid is replaced by FBS, the number of the extracted cell nuclei is reduced, and the whole cell nuclei accounts for 45 percent;

DPBS in the cell nucleus purifying liquid is replaced by NF water, the number of the extracted cell nuclei is reduced, and the whole cell nuclei accounts for 20%.

From the above results, the components and ratios in the kit of example 1 cannot be optionally replaced or adjusted, which would result in a serious decrease in the number of extracted nuclei and a serious decrease in the total nuclei ratio.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (9)

1. The animal adipose tissue cell nucleus extraction kit is characterized by comprising a cell nucleus lysate, wherein the lysate contains 0.3-1 mM gradient centrifugation reagent, 30-100 mM citric acid, 0.1-1 mM DTT and 0.1-1U/mu L RNase inhibitor, and is prepared by nuclease-free water.

2. The adipose tissue cell nucleus extraction kit of claim 1, wherein the gradient centrifugation reagent comprises sucrose.

3. The adipose tissue cell nucleus extraction kit according to claim 1, wherein the concentration of citric acid is 30-60 mm.

4. The kit for extracting adipose tissue nuclei according to claim 1, further comprising a nuclear purification solution, wherein the nuclear purification solution contains 0.2-2% BSA, 0.5-2 mm DTT, and 1-3 u/μl rnase inhibitor, and is formulated with DPBS.

5. The method for preparing the kit for extracting the nuclei of the adipose tissue of the animal according to claim 1, wherein the gradient centrifugation reagent and the citric acid are added into the water without the nuclease according to the proportion, and the DTT and the RNase inhibitor are added before the kit is used.

6. An animal adipose tissue cell nucleus extraction method is characterized by comprising the following steps:

s1, obtaining 1-2 cm ³ Animal adipose tissue;

s2, transferring the animal adipose tissue obtained in the step S1 into a centrifuge tube, adding 1mL of the cell nucleus lysate of claim 1, and shearing to 1-2 mm ³ Size of the material;

s3, adding 1-3 mL of the cell nucleus lysate, and transferring to a homogenizer for grinding;

s4, adding 1-3 mL of the cell nucleus lysate, blowing, uniformly mixing, sieving, centrifuging for 3-10 min at 2-6 ℃ and 300-800 g, and removing the supernatant to obtain the animal adipose tissue cell nucleus.

7. The method for extracting adipose tissue nuclei according to claim 6, further comprising:

s5, re-suspending the animal adipose tissue cell nucleus obtained in the step S4 by using the cell nucleus purification liquid of claim 4, centrifuging at 2-6 ℃ for 3-10 min at 300-800 g, removing supernatant, and re-suspending by using the cell nucleus purification liquid to obtain the purified animal adipose tissue cell nucleus.

8. The method according to claim 6 or 7, wherein in step S3, 6 μl of the polishing liquid is used in the polishing process, and the amount of released nuclei and the morphology of nuclei are observed by microscopic examination after staining with trypan blue, and polishing is stopped when the cell concentration is more than 200 nuclei/μl.

9. The method of claim 6 or 7, wherein the animal is a mouse.