阅读说明：本技术 一种单细胞测序筛选软骨细胞标记分子的方法 (Method for screening chondrocyte marker molecules by single cell sequencing ) 是由 于腾波 陈进利 赵海波 赵夏 姜弘元 王天瑞 马金龙 孙泽文 于承浩 李天予 韩 于 2021-03-08 设计创作，主要内容包括：本发明公开了一种利用单细胞测序筛选软骨细胞标记分子的方法,包括以下步骤：(1)分离软骨组织；(2)分离单细胞并进行裂解；(3)对样品的mRNA进行逆转录得到cDNA；(4)以cDNA为模板进行PCR扩增,产物纯化,进行Tagmentation反应,完成cDNA测序文库构建；(5)对构建的cDNA文库进行高通量测序；(6)测序结果进行生物信息学分析,寻找软骨细胞特异性的标记分子。(The invention discloses a method for screening chondrocyte marker molecules by single cell sequencing, which comprises the following steps: (1) separating cartilage tissue; (2) separating single cells and performing lysis; (3) carrying out reverse transcription on mRNA of a sample to obtain cDNA; (4) carrying out PCR amplification by taking cDNA as a template, purifying a product, and carrying out a mutagenesis reaction to complete the construction of a cDNA sequencing library; (5) carrying out high-throughput sequencing on the constructed cDNA library; (6) and (5) performing bioinformatics analysis on the sequencing result, and searching for a chondrocyte specific marker molecule.)

1. A method for screening chondrocyte marker molecules by single cell sequencing is characterized in that: the method comprises the following steps:

1) extracting cartilage tissue;

2) single cells were isolated and lysed: separating single cells, adding the single cells into a lysis buffer solution for lysis to obtain a cell lysate;

3) reverse transcription of mRNA from the sample to give cDNA: constructing a reverse transcription system by using a SuperScript kit of ThermoFisher company in America, and performing reverse transcription on the cell lysate obtained in the step 2) to obtain cDNA;

4) and (3) carrying out PCR amplification by taking the cDNA as a template to construct a sequencing library, and sequencing: amplifying and purifying the reverse transcription product obtained in the step 3) by using a NexteraXT kit of the U.S. Illumina company to construct a high-throughput sequencing library; after completion, the library is submitted for sequencing;

5) and (4) performing bioinformatics analysis on the sequencing result, and searching for a chondrocyte specific marker molecule.

2. The method for screening chondrocyte marker molecules by single cell sequencing according to claim 1, wherein: in the step 1), single cell sequencing is carried out on chondrocytes extracted from cartilage tissues, and chondrocyte-specific marker molecules are searched from an mRNA level.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for screening chondrocyte marker molecules by single cell sequencing.

Background

Osteoarthritis is a chronic degenerative joint disease in which articular cartilage is degraded, subchondral bone is hardened, osteophytes are formed with joint pain, swelling, deformity and movement disorder, and is often found in load-bearing joints such as knees and marrow. The worldwide osteoarthritis prevalence rate is about 15% according to statistics, wherein the incidence rate of the elderly people over 50 years old reaches 50%, and the final disability rate reaches 53%. The national epidemic investigation and research shows that the total disease rate of knee, hand, neck and waist arthritis of people over 40 years old in China is as high as 46.3 percent. The loss and disability of joint function not only seriously affects the life quality of the old people, but also increases the economic burden of families and society, and effectively prevents the disease, which becomes a great public health problem at home and abroad.

Osteoarthritis is closely related to dysbalance of synthesis and degradation of cartilage extracellular matrix caused by accumulation of aged chondrocytes, decomposition and degeneration of joint cartilage tissues and continuous pathological inflammatory state in joints. The phenomenon of aging often occurs in articular chondrocytes of patients with clinical osteoarthritis, and the phenomenon becomes more pronounced as the articular cartilage tissue deteriorates. Chondrocyte senescence plays an important role in the development of osteoarthritis, so that delaying or eliminating senescent chondrocytes has the potential to inhibit the inflammatory response of osteoarthritis, protect the integrity of articular cartilage tissue, improve the pathological changes of osteoarthritis, and relieve pain of osteoarthritis patients.

Therefore, if specific marker molecules and regulatory factors of chondrocytes can be continuously discovered, and the interaction between osteoarthritis and chondrocytes is discussed from a molecular level, a more effective scientific basis can be provided for deeply disclosing the pathogenesis of osteoarthritis, and a new thought and a new method can be provided for clinically treating osteoarthritis.

The whole transcriptome is amplified and sequenced on the single cell level of the chondrocytes by using a single cell sequencing technology, the gene transcription condition and the transcription regulation rule in the cells are researched on the whole level, and finally, the specific marker molecules of the chondrocytes are analyzed and searched on the mRNA level by using a bioinformatics analysis technology, so that a new target point is provided for targeted therapy of osteoarthritis.

Disclosure of Invention

The invention aims to provide a method for screening chondrocyte marker molecules by single cell sequencing aiming at the existing scientific problems.

The technical scheme of the invention is summarized as follows:

the method for screening the chondrocyte marker molecules by single cell sequencing comprises the following steps:

1) extracting cartilage tissue;

2) single cells were isolated and lysed: separating single cells, adding the single cells into a lysis buffer solution for lysis to obtain a cell lysate;

3) reverse transcription of mRNA from the sample to give cDNA: constructing a reverse transcription system by using a SuperScript kit of ThermoFisher company in America, and performing reverse transcription on the cell lysate obtained in the step 2) to obtain cDNA;

4) and (3) carrying out PCR amplification by taking the cDNA as a template to construct a sequencing library, and sequencing: amplifying and purifying the reverse transcription product obtained in the step 3) by using a NexteraXT kit of the U.S. Illumina company according to the instruction to construct a high-throughput sequencing library; after completion, the library is submitted for sequencing;

5) and performing bioinformatics analysis on the sequencing result, and analyzing from the mRNA level to search for a chondrocyte specific marker molecule.

In the step 1), single cell sequencing is performed on the cells extracted from the cartilage tissue, and the specific marker molecules are searched from the mRNA level by bioinformatics analysis.

The outstanding substantive features and remarkable progress of the invention are as follows:

by using a single cell sequencing technology, the single cell sequencing is carried out on the chondrocytes extracted from the cartilage tissues, and the bioinformatics analysis is used for searching specific marker molecules of the chondrocytes from the mRNA level, so that a more comprehensive, objective and accurate method is provided for searching new targets for targeted therapy.

Drawings

FIG. 1 shows single cell sequencing to detect cell subsets in cartilage tissue.

FIG. 2 is a bubble chart showing the expression of marker molecules in chondrocytes in cartilage tissue.

FIG. 3 is a graph showing marker molecule expression of chondrocytes in cartilage tissue using clustered heat.

Detailed Description

The following is a detailed description of the implementation operation of the present invention, and the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following implementation examples.

The present invention will be further described with reference to the following examples.

1. High-quality cartilage histiocyte is obtained by using a self-created extraction method.

2. A single cell sample of cartilage tissue was prepared. Single cells were isolated and lysed: the single cells were isolated and lysed by adding lysis buffer.

3. Reverse transcription of mRNA from the sample to give cDNA: constructing a reverse transcription system by using a SuperScript kit of Thermo Fisher company in USA, and performing reverse transcription on the cell lysate obtained in the step 3) to obtain cDNA.

4. Amplifying by taking cDNA as a template, constructing a sequencing library, sequencing, amplifying and purifying the reverse transcription product obtained in the step 4) by using a Nextera XT kit of the U.S. Illumina company according to the instruction, and constructing a high-throughput sequencing library; after completion the library was submitted for sequencing.

5. The chondrocyte-specific marker molecules were searched for by bioinformatic analysis.

Example 1

In this example, knee articular cartilage of osteoarthritis patients in clinical surgery was selected as the experimental material, and chondrocytes thereof were subjected to sequencing of single-cell transcriptome.

1. Obtaining chondrocytes in cartilage tissue:

1) the extraction method of the chondrocytes comprises the following steps:

s1) cutting cartilage tissue by using an ophthalmic scissors to obtain a single cell;

s2) sorting the single cells to obtain the chondrocytes.

The experiment is repeated three times, and the specific steps of each repeated experiment are as follows:

2) preparation of Single cells

The cartilage tissue was carefully dissected from the sterile knee joints of the patients within the operating table using sterile scissors and sterile forceps. The cartilage tissue was slightly cut with an ophthalmic scissors and soaked in 0.01M PBS for 2min, and then taken out, washed with PBS, and washed 3 times to obtain the soaked cartilage tissue. And (3) fully shearing the soaked cartilage tissue on a sterile plate by using an ophthalmic scissors, and dripping 0.01M PBS (phosphate buffer solution) at proper time in the process of shearing the soaked cartilage tissue to keep the cartilage tissue in a wet state to obtain the sheared cartilage tissue. And (3) adding the collagenase I solution into the sterile plate containing the cut cartilage tissue, and blowing the cartilage tissue by using a 1ml head which is cut to be over, so as to obtain a collagenase I and cartilage tissue mixture.

(1) And (3) vortex: shaking a 50ml centrifuge tube containing collagenase I and cartilage tissue mixture on a shaker for 3min to obtain a shaken collagenase and cartilage tissue mixture, digesting in an incubator for 30min, taking out the mixture every 5min, and vortexing

(2) The collagenase is stopped with complete medium, left to stand for 2min, centrifuged for 300g (rcf),10min and washed 2 times with PBS.

(3) The resulting cells were incubated on ice for antibodies: 10⁷cell + 100. mu. Lbuffer + 10. mu.L antibody, protected from light, shaken well, incubated for 10min in a refrigerator at 2-8 ℃.

(4)10⁷cell +2mlb buffer wash, 300g (rcf)10 min.

(5) After centrifugation, every 10⁷Adding 80 μ L buffer to the cell, 10 times⁷The cell was mixed with 20. mu.L of magnetic beads and incubated at 2-8 ℃ for 15min in a refrigerator.

(6) Every 10 th⁷2mL buffer was added to the cells, and 500. mu.Lb buffer was added thereto after 10min of washing with 300g (rcf). LS type column was wetted with 3ml buffer, the cell suspension was applied to the column and the column was passed through the column and washed 3 times separately after the completion of the dropping (3 ml each time of LS column).

(7) Cells were harvested by quickly removing the LS column on the shelf and adding 5ml buffer.

Culturing the chondrocytes under the following culture conditions: DMEM + 10% FBS + 1% double antibody. Adherent chondrocytes were obtained.

2. Single cells were isolated and lysed:

1) cleaning the worktable and the pipette tip by RNaseZap and a solution without DNA;

2) the following reagents were mixed as lysis buffer:

A) 19 μ l of 0.2% Triton-X100 solution;

B) 40U/. mu.l RNase inhibitor 1. mu.l.

3) Add 1.19. mu.l lysis buffer to a 0.2ml thin-walled PCR tube;

4) single cells were isolated from the cells obtained in step (2) in a minimum volume, typically 0.3. mu.l, and added to a PCR tube containing lysis buffer.

4. Reverse transcription of mRNA from the sample to give cDNA:

1) the following reagents were mixed as primer-dNTP premix:

A)dNTP(10mM)10μl；

B) Oligo-dT 30VN primer (10. mu.M) 10. mu.l.

2) The following reagents were mixed as a premix for reverse transcription:

A) SuperScript III first-strand buffer 2. mu.l from ThermoFisher, USA;

B) SuperScript III reverse transcriptase solution 0.5. mu.l from ThermoFisher, USA;

C) RNAse inhibitor (40U/. mu.l) 0.25. mu.l;

D) DTT solution (100mM) 0.5. mu.l;

E) betaine solution (5M) 2. mu.l;

F) 0.06. mu.l of magnesium chloride solution (1M);

G) TSO solution (100. mu.M) 0.2. mu.l;

H) ERCC exogenous RNA standard sample solution 0.5. mu.l.

3) Adding 2 mu l of primer-dNTP premix into the PCR tube containing the cells and the lysis buffer obtained in the step (3);

4) quickly vortex and shake the PCR tube to fully mix the solution, and immediately place the solution on ice after centrifuging the solution for 10s at 700g at room temperature;

5) incubate the sample at 72 ℃ for 3min and immediately return to ice;

6) centrifuging at 700g for 10s at room temperature, collecting the liquid at the bottom of the PCR tube, and placing the liquid on ice;

7) adding the reverse transcription premixed solution into the single cell lysate, gently blowing and sucking the sample for several times by using a pipettor, and avoiding generating bubbles in the process of mixing;

8) centrifuging at 700g for 10s at room temperature, and collecting liquid at the bottom of the PCR tube;

9) and (3) placing the PCR tube containing the sample in a PCR instrument, and setting a program to perform reverse transcription of cell mRNA to obtain a cDNA first strand reaction product.

4. And (3) amplifying by taking the cDNA as a template to construct a sequencing library, and sequencing:

1) the following solutions were mixed as a PCR mixture:

A) 10 mu l of first chain reaction product obtained in the step (4);

B) 12.5. mu.l of KAPA HiFiHotStartStreammix solution from Roche Molecular Systems, USA;

C) ISPCR primer solution (10. mu.M) 0.25. mu.l;

D) nuclease-free water 2.25. mu.l.

2) Adding 15 mu l of the PCR mixed solution obtained in the step 1) into a PCR tube, mixing the sample by vortex oscillation, centrifuging the mixture for 10s at 700g at room temperature, and collecting the liquid at the bottom of the PCR tube;

3) the PCR tube containing the sample was placed in a PCR instrument and the cell cDNA was programmed to amplify.

4) Placing AMPure XP magnetic beads of American Beckman Coulter company for 15min at room temperature, and vortexing, shaking and uniformly mixing;

5) adding 25 mu l of magnetic beads into the cDNA amplification product obtained in the isometric step 3), sucking by a pipette for 10 times, uniformly mixing, transferring the solution into a 96-well plate, and incubating for 8min at room temperature;

6) placing 96-well plate on magnetic frame for 5min, and carefully removing supernatant when the solution is clear; the beads were washed with 200. mu.l of 80% ethanol, incubated for 30s and carefully removed ethanol, repeated 2 times;

7) after completely removing the ethanol, standing at room temperature for 5min, adding 15 μ l of EB solution after a crack appears on the surface of the magnetic bead, and sucking by a pipette for 10 times to mix uniformly;

8) removing the magnetic frame, and standing at room temperature for 2 min; putting the 96-well plate back to the magnetic frame again, standing for 2 minutes, sucking 13 mu l of supernatant after the solution is clarified, and transferring the supernatant into a new 0.2ml thin-wall PCR tube;

9) detecting the fragment size distribution of the cDNA amplification product by using 6% PAGE gel, wherein the fragment size of a good library should be 500-3000 bp, no short fragment smaller than 500bp exists, and a peak should appear between 1500-2000 bp;

10) the following solutions were mixed as the labeled premix:

A) 4. mu.l of labeled DNA buffer from Illumina, USA;

B) 1.67. mu.l of labeled enzyme mixture from Illumina, USA;

C) resuspension buffer 1.33. mu.l from Illumina USA.

11) Taking 1 μ l of the cDNA obtained in the step 8), adding 7 μ l of the labeled premix obtained in the step 10), incubating at 55 ℃ for 10min, and labeling the cDNA;

12) then 2 mul of NT buffer solution of the American Illumina company is added, and the incubation is carried out for 5min at room temperature, thus finishing the labeling reaction;

13) the following solutions were mixed as a pre-mix for adaptor ligation amplification:

A) 15 μ l of KAPA HiFiHotStartStreacMix solution from Roche Molecular Systems, USA;

B) 2.5. mu.l of Index 1 primer solution from Illumina, USA;

C) 2.5. mu.l of Index 2 primer solution from Illumina, USA.

14) Mu.l of the adaptor ligation premix obtained in step 13) was added to 10. mu.l of the labeled product obtained in step 12), and the mixture was placed in a PCR apparatus and the procedure was set to ligate the adaptors.

15) Placing AMPure XP magnetic beads of American Beckman Coulter company for 15min at room temperature, and vortexing, shaking and uniformly mixing;

16) mu.l of the beads were mixed as 0.8: adding the volume ratio of 1) into 30 mu l of the product obtained in the step 13), sucking by a pipette for 10 times, uniformly mixing, transferring the solution into a 96-well plate, and incubating for 8min at room temperature;

17) repeating the steps 6) to 8), and purifying the amplification product;

18) fragment size distribution of the sequencing libraries was obtained using 6% PAGE gels and 2100Bioanalyzer detection step 17) from Agilent, USA, and the concentration of each library DNA was measured using a Qubit3.0 fluorometer from ThermoFisher, USA;

19) calculating the molar ratio of each library in the final library mixed solution according to the concentration of each library measured in the step 18) and the data amount required to be sequenced, mixing the libraries in proportion, measuring by using a Qubit3.0 fluorimeter, and adjusting the final concentration to 5 nM;

20) sent to sequencing company for high throughput sequencing.

The cartilage tissue of osteoarthritis patients is selected as the experimental material only for example, which proves that the method can be successfully applied to the sequencing of the single cell transcriptome of the human articular cartilage tissue, but the invention can also be applied to the sequencing of the single cell transcriptome of human and other related cartilage cells.

The invention is not limited to the specific embodiments described above, but extends to any novel feature or novel combination disclosed herein, or to any novel method or process step or novel combination disclosed.