CN115466714A - Low-temperature dissociation method suitable for mouse liver single cell sequencing - Google Patents
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Abstract
The invention discloses a low-temperature dissociation method suitable for mouse liver single cell sequencing, which comprises the steps of harvesting fresh liver tissues for processing; then placing the mixture into a culture dish added with the bacillus licheniformis protease mixture for dissociation; and incubating at 2-6 deg.C to obtain tissue homogenate; screening the tissue homogenate by a cell sieve, washing a filter by using a DMEM or 1640 culture medium, and stopping enzyme activity to obtain a cell suspension; centrifuging the cell suspension, and removing a supernatant; resuspending the cells in DMEM or 1640 culture medium containing fetal calf serum, and sieving the cells; collecting cell sediment, and using DPBS to re-suspend cells to obtain dissociated liver single cells. The low-temperature dissociation method suitable for mouse liver single cell sequencing provided by the invention solves the problems of parenchymal hepatic cell death, a large amount of stress gene expression in the retained cells and the like caused by the traditional thermal dissociation experiment method. The method has better technical performance, is convenient and effective, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a low-temperature dissociation method suitable for mouse liver single cell sequencing.
Background
The preparation of single cell suspension is a key experimental step for carrying out single cell sequencing on solid tissues, when digestive enzymes are selected for preparing the single cell suspension, the enzymolysis temperature, the enzyme activity intensity, the enzyme concentration and the incubation time are the most important factors, and the fluctuation of the cell activity, the cell yield, the cell integrity and the gene expression integrity can be caused by the change of the factors. The current widely used experimental method is a "thermal dissociation" method, i.e. tissue dissociation is carried out under incubation conditions of 30-37 ℃. Researchers have come to focus on the problems that can arise with "thermal dissociation" approaches, such as causing gene expression Artifacts (Artifacts) (Adam M, et al.2017); inducing the unnatural expression of immediate early response genes (IEGs), such as Fos, jun or other activator protein-related genes, transcription factors or Heat Shock Proteins (HSPs) (Denisenko E, et al 2020); loss of some cell types, such as neurons and astrocytes (Mattei D, et al 2020); altering the expression pattern of cell surface markers, etc. (Mattei D, et al 2020). Particularly, in liver tissues, since liver parenchymal cells are very poor in enzyme tolerance at a hot temperature, many liver parenchymal cells die in a conventional liver dissociation method, so that single cell sequencing of liver cannot capture liver parenchymal cells, and a large amount of stress genes are expressed in remaining cells. Based on the above background, it is necessary to establish a new tissue dissociation experimental method to solve the problems caused by the "thermal dissociation" method.
At present, no mature and effective tissue dissociation method exists, the problems of gene expression disturbance, stress gene unnatural state expression, dissociation cell preference and the like in cell suspension are solved. The existing dissociation enzymes, such as trypsin, collagenase, neutral protease and the like, all play the highest enzymolysis efficiency at about 30 ℃ and do not avoid the problem of thermal stimulation. Lower incubation temperatures reduce the speed of the enzyme reaction and thus prolong the enzymatic hydrolysis time, but low temperature incubation can reduce cell death and reduce the irritation of high temperature incubation on the cells.
Disclosure of Invention
Aiming at the technical problems existing at present, the invention aims to provide a low-temperature dissociation method suitable for mouse liver single cell sequencing.
The low-temperature dissociation method based on mouse liver single cell sequencing provided by the invention has better performance for solving the problems. The solution of "cold dissociation" can make the transcription activity of the cell in a dormant state at 4 ℃ or lower, so that the gene expression in the cell is "frozen", thereby solving the problems of cell death and gene expression mode change caused by the prior thermal dissociation environment. The cold dissociation requires that the enzyme still keeps higher activity under the condition of low temperature, and the protease purified from psychrophilic microorganisms (serine protease separated from Himalayan glacien resident bacteria bacillus licheniformis, bacillus licheniformis protease A) meets the requirement of the cold dissociation on the enzyme.
In order to achieve the purpose, the invention adopts the following technical means:
a low-temperature dissociation method suitable for mouse liver single cell sequencing comprises the following steps:
s1, harvesting fresh liver tissues, and perfusing precooled 1x PBS through portal vein to make blood in the tissues flow out along with perfusate; wherein PBS is precooled at 4 ℃ and the filling amount is 3-5mL;
s2, continuously filling the precooled bacillus licheniformis protease mixture through the portal vein; wherein the Bacillus licheniformis protease mixture is prepared by dissolving Bacillus licheniformis protease A (VIII type) in phosphate buffer solution (DPBS) containing no calcium and magnesium ions, and has a concentration of 100mg/mL and a pH of 8.0; precooling the bacillus licheniformis protease mixture at 4 ℃ and filling the mixture in an amount of 3-5mL;
s3, taking out the perfused liver tissue, putting the liver tissue into a culture dish added with a bacillus licheniformis protease mixture, and cutting the liver tissue into pieces by using a blade to obtain a dissociation mixed solution; in the whole process, the culture dish is placed on ice at 4 ℃;
s4, transferring the dissociation mixed solution obtained in the step S3 into a container, and incubating at the temperature of 2-4 ℃ to obtain tissue homogenate; during the incubation period, the large tissue pieces can be cut up again by a blade, the dosage of the bacillus licheniformis protease mixture can be increased, and the incubation time can be increased, so that the liver tissue is fully contacted with the bacillus licheniformis protease mixture, and the maximum number of cells are released;
s5, screening the tissue homogenate by a cell sieve, washing the filter by a DMEM or 1640 culture medium, and stopping the activity of the enzyme to obtain a cell suspension;
s6, centrifuging the cell suspension, and removing a supernatant; resuspending the cells in DMEM or 1640 culture medium containing fetal calf serum, and sieving the cells;
and S7, collecting cell precipitates, and using DPBS to resuspend cells to obtain dissociated single liver cells.
Further, the specific process of step S4 is as follows: transferring the liver tissue obtained in the step S3 into a 5mL centrifuge tube with a cover, shaking and incubating at the temperature of 4 ℃, and keeping the incubation time for 1 hour to obtain tissue homogenate; the large pieces of tissue may be re-minced during incubation with a razor blade to ensure that the liver tissue is sufficiently exposed to the bacillus licheniformis protease mixture to release the maximum number of cells;
preferably, the Bacillus licheniformis protease is purchased from Bacillus licheniformis enzyme (Catalog # P5380) from Sigma.
Preferably, in step S5, the DMEM is DMEM containing 20wt% fetal bovine serum;
preferably, in step S6, the DMEM containing fetal bovine serum is DMEM containing 10% fetal bovine serum;
preferably, in step S7, the DPBS is a DPBS containing 1wt% BSA.
Further, the low-temperature dissociation method further comprises the step of measuring the concentration and counting by using a blood cell counting plate or a Countess II automatic cell counter, wherein the concentration measured by the blood cell counting plate or the Countess II automatic cell counter ranges from 700 to 1200nuclei/ul.
Further, the application of the single liver cell prepared by the low-temperature dissociation method comprises the following steps:
s101, cell suspension is captured through a 10x Genomics single cell platform, and the transcript marking, reverse transcription and library construction in the nucleus are carried out
S102, after the library construction is finished, firstly using the qubit3.0 to carry out preliminary quantification, diluting the library to 1ng/ul, then using Agilent 2100 to detect the insert size of the library, and after the insert size meets the expectation, using a Bio-RAD CFX 96 fluorescence quantitative PCR instrument, bio-Performing Q-PCR on RAD KIT iQ SYBRGRN, and accurately quantifying the effective concentration of the library, wherein the effective concentration of the library is more than 10nM so as to ensure the quality of the library. Quality-qualified library Illumina platform (Illumina Novaseq) TM 6000 ) was sequenced. The sequencing strategy was PE150.
S103, data analysis is carried out based on CellRanger and Seurat software.
The invention also provides an application of the low-temperature dissociation method suitable for mouse liver single cell sequencing in preparation of single cells of other solid tissues such as kidney, heart, brain and the like.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the low-temperature dissociation method suitable for mouse liver single cell sequencing provided by the invention solves the problems of parenchymal hepatic cell death, a large amount of stress gene expression in the retained cells and the like caused by the traditional thermal dissociation experiment method. The method has better technical performance, is convenient and effective, and is suitable for popularization and application.
Drawings
FIG. 1 shows acridine orange/propidium iodide (AO/PI) staining patterns of mouse liver cell suspensions of example 1 of the present invention;
FIG. 2 is a graph showing the result of quality control of mouse liver single cell sequencing data analysis in example 1 of the present invention;
FIG. 3 is a graph showing the result of cell identification by sequencing data analysis of mouse liver single cells in example 1 of the present invention;
FIG. 4 shows the cell types detected by "cold dissociation" of example 1 of the present invention and "thermal dissociation" of comparative example 1;
FIG. 5 shows the stress gene expression ratios measured by "cold dissociation" in example 1 of the present invention and "thermal dissociation" in comparative example 1.
Detailed Description
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety and their equivalent family patents are also incorporated by reference, especially as they disclose definitions relating to synthetic techniques, products and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing numerical values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), 1 unit is considered to be 0.0001,0.001,0.01 or 0.1, as appropriate. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed 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.
When used with respect to chemical compounds, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively) unless expressly specified otherwise. In addition, unless explicitly stated otherwise, the use of the terms "a", "an" or "the" are intended to include the plural forms as well.
The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not relevant to whether such other component, step or procedure is disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of … …" excludes any other components, steps or processes from the scope of any such term as are hereinafter recited, out of those necessary for performance. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in 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 and the disclosures and references cited herein and the materials to which they refer are incorporated 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 following claims.
Example 1
A C57BL/6 mouse of 7 weeks old is anesthetized to take a fresh liver sample, and the embodiment of the invention is adopted to carry out low-temperature dissociation and single-cell sequencing experiments on liver tissues. The specific embodiment is as follows:
s1, perfusing 4mL of 4 ℃ precooled 1xPBS into harvested fresh mouse liver tissues through portal veins to enable blood in the tissues to flow out completely along with perfusate;
s2, continuously perfusing 4mL of a 4 ℃ pre-cooled 1mg/mL Bacillus licheniformis protease mixture (Sigma Catalog # P5380) into liver tissue through the portal vein;
s3, taking out the perfused liver tissue, putting the liver tissue into a culture dish added with 1mL of the bacillus licheniformis protease mixture, and thoroughly cutting the liver tissue into small pieces by a blade; in the whole process, the culture dish is placed on ice at 4 ℃;
s4, transferring the dissociation mixed solution into a 4mL centrifuge tube with a cover, shaking and incubating at the temperature of 4 ℃, and keeping the incubation time for 1 hour to obtain tissue homogenate;
s5, screening the tissue homogenate by a 70-micron cell sieve, washing a filter by a DMEM medium containing 20% fetal calf serum, and stopping enzyme activity to obtain a cell suspension;
s6, centrifuging 250g of cell suspension at 4 ℃ for 5min, and removing supernatant; resuspending the cells in DMEM containing 10wt% fetal calf serum and then sieving through a 40 μm cell sieve;
s7, collecting cell pellets, resuspending the cells using DPBS containing 1wt% BSA, and placing on ice for use; and stained with acridine orange/propidium iodide, the results are shown in FIG. 1;
s8, measuring the concentration and counting by adopting a Countess II automatic cell counting instrument, wherein the cell concentration is 1035 nucleoli/ul, and the total cell amount is 8.42 ten thousand;
subsequently, the cell nucleus suspension was subjected to capture and in-cell transcript labeling, reverse transcription, and library construction (according to the 10x Genomics labeling and library construction experimental procedure) by a 10x Genomics single cell platform, expecting 1 ten thousand nuclei to be captured; after the library construction is completed, illumina Novaseq is adopted TM 6000, sequencing, wherein the sequencing strategy is PE150, and the predicted output data volume is 100G Base; data analysis was performed based on cellrange and seruat software, and the results are shown in figure 2 quality control results and figure 3 cell identification results.
Comparative example 1
Obtaining liver single cells from the harvested fresh mouse liver tissues by using a traditional thermal dissociation method, which comprises the following steps:
1. shearing the harvested fresh mouse liver tissues into small pieces, and putting the small pieces into a screw pipe without nuclease;
2. preparing collagenase II (1.5 mg/mL), neutral protease (1 mg/mL), papain (20U/mL) and deoxyribonuclease I (10 mg/mL), adding the mixed enzyme into the threaded tube, and incubating for 10min in a shaking table at 30 ℃ in a constant-temperature water bath;
3. passing the digestive juice through a 70-micron cell filter, transferring the filtrate into a 50mL centrifuge tube, centrifuging at 4 ℃ for 5 minutes at 500 g;
4. adding 1mL of erythrocyte lysis buffer solution into the precipitate, blowing, resuspending, placing on ice for 5min, and stirring regularly;
5. adding 9mL phosphate buffer solution precooled at 4 ℃, then centrifuging for 5 minutes at 500g at 4 ℃, sucking out supernate and discarding, and keeping precipitates;
6. adding 5mL of precooled phosphate buffer solution at 4 ℃ containing 0.04% of fetal bovine serum albumin, and suspending the precipitate;
7. then, the filtrate is transferred to a 50mL centrifuge tube through a 30um cell filter to obtain the mouse liver single cell suspension.
Experiment one:
the cell types and stress gene expression of the single liver cells prepared in example 1 and comparative example 1 were examined, and the results are shown in fig. 4 and fig. 5, respectively.
As can be seen from fig. 4 and 5, the single cells obtained by cold dissociation had fewer cell types and significantly lower stress gene expression.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (6)
1. A low-temperature dissociation method suitable for mouse liver single cell sequencing is characterized by comprising the following steps:
s1, harvesting fresh liver tissues, and perfusing precooled 1xPBS through portal vein to make blood in the tissues flow out along with perfusate;
s2, continuously filling the precooled bacillus licheniformis protease mixture through the portal vein;
s3, taking out the perfused liver tissue, putting the liver tissue into a culture dish added with a bacillus licheniformis protease mixture, and cutting the liver tissue into pieces by a blade to obtain a dissociation mixed solution; in the whole process, the culture dish is placed on ice at 4 ℃;
s4, transferring the dissociation mixed solution obtained in the step S3 into a container, and incubating at the temperature of 2-4 ℃ to obtain tissue homogenate;
s5, screening the tissue homogenate by a cell sieve, washing the filter by a DMEM or 1640 culture medium, and stopping the activity of the enzyme to obtain a cell suspension;
s6, centrifuging the cell suspension, and removing a supernatant; resuspending the cells in DMEM or 1640 culture medium containing fetal calf serum, and sieving the cells;
and S7, collecting cell precipitates, and using DPBS to resuspend cells to obtain dissociated single liver cells.
2. The low-temperature dissociation method suitable for mouse liver single cell sequencing of claim 1, wherein the specific process of step S4 is as follows: and (4) transferring the liver tissue obtained in the step (S3) into a 5mL centrifuge tube with a cover, shaking and incubating at the temperature of 4 ℃, and keeping the incubation time for 1 hour to obtain tissue homogenate.
3. The cryodissociation method for sequencing single cells of mouse liver according to claim 1, wherein the DMEM is DMEM containing 20wt% fetal bovine serum in step S5.
4. The cryodissociation method for sequencing single cells of mouse liver according to claim 1, wherein in step S6, the DMEM containing fetal bovine serum is DMEM containing 10% fetal bovine serum.
5. The method of claim 1, wherein in step S7 the DPBS is DPBS comprising 1wt% BSA.
6. The cryodissociation method for mouse liver single cell sequencing according to claim 1, wherein the cryodissociation method further comprises the step of measuring the concentration of the cell suspension by using a blood cell counting plate or a Countess II automatic cell counter, and counting the concentration, wherein the concentration measured by the blood cell counting plate or the Countess II automatic cell counter ranges from 700 to 1200 nucleolei/ul.
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