CN112280828A - In vitro tissue cell nucleus separation method for reducing single cell amplification bias - Google Patents
In vitro tissue cell nucleus separation method for reducing single cell amplification bias Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a method for separating cell nucleuses by single cell sequencing for reducing amplification bias and application thereof, wherein the method comprises the following steps: carrying out cracking treatment on a sample to be extracted in a second buffer solution; carrying out first centrifugal treatment on the lysate to obtain cell nucleus sediment; and washing and resuspending the cell nucleus precipitate in a first buffer solution, performing second centrifugation treatment, and finally, uniformly resuspending the cell nucleus precipitate in a third buffer solution to obtain the finally required cell nucleus. The method can obtain sufficient and complete single cell nucleuses from fewer tissue samples or cells for single cell whole gene experimental research by only using conventional and simple reagent consumables without expensive separation equipment, long-time gradient centrifugation and other operations.
Description
Technical Field
The invention relates to the field of gene sequencing, and relates to an in vitro tissue cell nucleus separation method suitable for reducing single cell amplification bias.
Background
Single cell sequencing refers to a new technology for performing high-throughput sequencing analysis on multiple groups of genomes, transcriptomes, epigenets and the like on a single cell level. The application of the technology makes it possible to explain the gene structure and gene expression state of single cells and to interpret the heterogeneity between cells. The single cell sequencing can be widely applied to researches in developmental biology, immunity, tumor and other directions, and a large number of high-quality researches and scientific achievements are shown. The single cell whole genome sequencing is to perform whole genome amplification on DNA of single cells obtained after dissociation in tumor tissues, then perform library construction and sequencing, analyze the gene condition in each cell, study the tumor occurrence and development mechanism, molecular typing and the like, and provide a new direction for tumor typing, tumor treatment strategies, new drug research and development and the like.
Single cell sequencing has high requirements on sample activity. Human tumor tissues have multiple necrotic lesions, high-survival rate single cell suspension is not easy to obtain, and the cell survival rate cannot be ensured in the transportation process of the single cell suspension. The existing realization method is to carry single cell separation equipment to a hospital, and the fresh excised tissue cut by an operation is immediately digested and subsequently separated, so that the experiment is greatly limited in cost, time, place and other aspects. Therefore, there is a need to develop a new sample processing method to more conveniently obtain a suitable sample for single cell whole genome sequencing.
Fresh in-vitro tumor tissues are frozen into frozen tissues by liquid nitrogen and then can be transported and stored by dry ice, but the frozen tissues are difficult to digest to obtain single cell suspension with high survival rate after being thawed. The rapid freezing and cryopreservation processes of fresh tissues can cause great damage to cells, RNA in the cells is free and lost, single-cell RNA sequencing cannot be performed, and nuclear membranes can be kept intact in the freezing and thawing processes. It has been shown that the nucleus contains sufficient amounts of genomic DNA for sequencing and is highly relevant for genome sequencing with whole cells. Since nuclear membranes are more easily kept intact than cell membranes, obtaining a suspension of nuclei from frozen tissue is more practical than obtaining a suspension of single cells. At present, a mature and stable method for obtaining the mononuclear suspension from the frozen tissue does not exist, only a method for separating the mononuclear suspension from the frozen brain tissue by expensive auxiliary equipment exists at present, and the demonstration of quality control results does not exist.
Meanwhile, a hospital sample library has a large number of special cases and rare frozen samples, and the utilization of the frozen samples can be greatly helpful for researching rare disease species and shortening the research period.
Therefore, it is urgent to develop a simple and convenient method for separating human in vitro frozen tumor tissue cell nucleus to obtain single cell nuclear suspension with high survival rate.
Disclosure of Invention
The present invention is intended to solve the technical problems in the related art at least to some extent: how to separate the in vitro frozen tumor tissue cell nucleus by a simple and convenient method. Therefore, the inventor develops a simple method, can obtain the mononuclear suspension suitable for single cell sequencing from the in vitro frozen tumor tissue sample, removes the time and place limitation of the single cell sequencing of the fresh in vitro tumor tissue, and enables the single cell sequencing technology to be more widely applied to the research of the in vitro tumor tissue microenvironment.
The first aspect of the present invention is a method for extracting cell nuclei, the method comprising the steps of:
1) performing mechanical sample cracking treatment in a buffer solution II (LB), and performing ice placement treatment and blowing treatment to obtain a cracking product;
2) performing first filtration treatment and first centrifugation treatment on the lysate to obtain a first centrifugation cell nucleus precipitate;
3) washing the first centrifugal cell nucleus sediment in a first buffer solution (WB), performing first resuspension treatment and second filtration treatment, and then performing second centrifugation treatment to obtain a second centrifugal cell nucleus sediment;
4) performing second suspension and uniform mixing treatment on the second centrifugal cell nucleus precipitate in a No. three (NB) buffer solution, and performing third filtration treatment to obtain a cell nucleus suspension;
further, the sample is an ex vivo sample.
Preferably, the ex vivo sample is ex vivo frozen tumor tissue or adherent cells cultured in vitro.
Further, buffer No. one (WB) includes: 10-20mM Tris-HCl (pH 7.5-7.8), 10-20mM NaCl, 50-100mM KCl, 2-10mM MgCl2，5-15mM CaCl20.04% BSA, 1mg/mL PI and 0.2U/. mu.L RNase inhibitor;
further, the mass-to-volume ratio of the sample to buffer one (WB) was 5mg:0.5 mL.
Further, buffer No. two (LB) includes: buffer No. one (WB) contained 0.2% NP-40 (by mass/volume).
Further, buffer No. three (NB) includes: D-Hanks buffer containing 1% FBS, 1mM DTT.
Further, buffer No. one (WB), buffer No. two (LB) and buffer No. three (NB) were pre-chilled at 4 ℃ before use.
Further, mechanical grinding and crushing treatment is carried out in the step 1) by using a grinding pestle.
Further, the volume of the sample after the crushing treatment in the step 1) is less than or equal to 0.2mm3。
Further, the ice-standing treatment time in the step 1) is 8-10min, and a liquid-transferring gun is used for uniformly mixing.
Further, the blow-beating treatment in the step 1) is 10-15 times.
Further, the first filtration treatment in step 2) was performed through a cell filter having a diameter of 70 μm.
Further, the first centrifugation treatment in step 2) was carried out at 500g at 4 ℃ for 5 min.
Further, the amount of buffer No. one (WB) used in the first resuspension treatment in step 3) was 0.5 mL.
Further, the second filtration treatment in step 3) was performed through a cell filter having a diameter of 40 μm.
Further, the filtrate obtained in the second filtration treatment in step 3) was collected in a new centrifuge tube and subjected to a second centrifugation treatment at 300g at 4 ℃ for 5 min.
Further, the third filtration treatment in step 4) was performed through a cell filter having a diameter of 40 m.
Further, in step 4), the second centrifuged cell nucleus pellet is subjected to a second resuspending and mixing treatment in 0.2mL of buffer III (NB).
Further, single cell whole gene amplification and amplification uniformity detection are carried out on the cell nucleus in the cell nucleus suspension.
Further, before single cell whole genome sequencing, the cell nuclei are further subjected to a resuspension treatment, and after the resuspension treatment, the concentration of the cell nuclei is 1000 nuclei/. mu.L.
In a second aspect, the present invention provides a method for extracting nuclei of in vitro frozen tumor tissue based on the method for extracting nuclei of the first aspect of the present invention, the method comprising:
step A1, pre-cooling the second buffer solution at 4 ℃, mechanically crushing the in-vitro frozen tumor tissue in the second buffer solution (LB), wherein the mass-volume ratio of the in-vitro frozen tumor tissue to the second buffer solution is not more than 5mg:0.5mL, and the volume of the in-vitro frozen tumor tissue after crushing treatment is not more than 0.2mm3Performing ice treatment on the mechanical crushing product for 5-10 min, and uniformly mixing by using a liquid transfer gun; then, blowing and beating the ice-setting processed product for 10-15 times to obtain a blown and beaten processed product;
step A2, performing first filtration treatment on the blow-beating treatment product obtained in the step A1, wherein the first filtration treatment is performed through a cell filter with the diameter of 70 microns, the filtrate after the first filtration treatment forms a lysate, and the lysate is subjected to first centrifugation treatment for 5min at the temperature of 4 ℃ and the weight of 500g to obtain cell nucleus sediment;
step A3, resuspending the cell nucleus sediment in a first buffer solution (WB), wherein the amount of the first buffer solution (WB) is 0.5mL, and the first resuspended cell nucleus suspension is formed by the resuspended first buffer solution (WB);
step A4, performing second filtration treatment on the first resuspended cell nucleus suspension, wherein the second filtration treatment is performed through a cell filter with the diameter of 40 μm, and the filtrate after the second filtration treatment forms second cell nucleus filtrate;
step A5, performing second centrifugation treatment on the second cell nucleus filtrate for 5min at the temperature of 4 ℃ and the weight of 300g to obtain cell nucleus precipitates after the second centrifugation treatment;
and step A6, re-suspending and uniformly mixing the cell nucleus sediment after the second centrifugal treatment by using 0.2mL of No. three buffer solution (NB) pre-cooled at 4 ℃, and then performing third filtration treatment to obtain the cell nucleus suspension of the in-vitro frozen tumor tissue.
Furthermore, before single-cell whole-gene amplification and amplification uniformity detection are carried out on the cell nuclei in the cell nucleus suspension of the in-vitro frozen tumor tissue, re-suspension treatment is carried out, and the concentration of the cell nuclei after the re-suspension treatment is 1000/mu L.
In a third aspect, the present invention provides a method for extracting nuclei from cultured adherent cells, based on the method for extracting nuclei of the first aspect of the present invention, the method comprising:
step B1, pre-cooling the second buffer solution at 4 ℃, mechanically crushing the cultured adherent cells in the second buffer solution (LB), wherein the mass-to-volume ratio of the cultured adherent cells to the second buffer solution is not more than 5mg:0.5mL, carrying out ice treatment on the mechanically crushed product for 5-10 min, and uniformly mixing by using a liquid transfer gun; then, blowing and beating the ice-setting processed product for 10-15 times to obtain a blown and beaten processed product;
step B2, performing first filtration treatment on the blow-beating treatment product obtained in the step A1, wherein the first filtration treatment is performed through a cell filter with the diameter of 70 mu m, the filtrate after the first filtration treatment forms a lysate, and the lysate is subjected to first centrifugation treatment for 5min at the temperature of 4 ℃ and the weight of 500g to obtain cell nucleus sediment;
step B3, resuspending the cell nucleus sediment in a first buffer solution (WB), wherein the amount of the first buffer solution (WB) is 0.5mL, and the first resuspended cell nucleus suspension is formed by the resuspended first buffer solution (WB);
step B4, performing second filtration treatment on the first resuspended cell nucleus suspension, wherein the second filtration treatment is performed through a cell filter with the diameter of 40 μm, and the filtrate after the second filtration treatment forms second cell nucleus filtrate;
step B5, performing second centrifugation treatment on the second cell nucleus filtrate for 5min at the temperature of 4 ℃ and at the weight of 300g to obtain cell nucleus precipitates after the second centrifugation treatment;
and step B6, re-suspending and uniformly mixing the cell nucleus sediment after the second centrifugal treatment by using 0.2mL of No. three buffer solution (NB) pre-cooled at 4 ℃, and then performing third filtration treatment to obtain the cell nucleus suspension of the cultured adherent cells.
Furthermore, before the cell nucleuses in the cell nucleus suspension of the cultured adherent cells are subjected to single cell whole gene amplification and amplification uniformity detection, the cell nucleuses are subjected to heavy suspension again, and the concentration of the cell nucleuses after the heavy suspension is 1000/mu L.
In a fourth aspect of the invention, there is provided a method of sequencing single cells based on the method of extracting nuclei of the first aspect of the invention, comprising the steps of:
step C1, extracting cell nuclei in the sample to be detected by using the cell nucleus extraction method of the first aspect of the invention;
and C2, performing single-cell whole genome sequencing on the cell nucleus so as to obtain a sequencing result.
In a fifth aspect of the invention, the invention provides an application of the cell nucleus obtained by the method for extracting the cell nucleus of the in-vitro frozen tumor tissue based on the second aspect of the invention in the research of tumor evolution mechanism, accurate cancer typing, tumor drug resistance mechanism and curative effect prediction after single-cell whole genome sequencing.
Has the advantages that:
1. the volume of the sample after the crushing treatment is less than or equal to 0.2mm3So as to lead the sample to be fully cracked and improve the recovery rate of nucleus and nucleus.
2. The presence of BSA in buffer No. two protects the integrity and discreteness of the nuclear membrane and prevents clumping.
3. The first centrifugal treatment is carried out for 5min at 4 ℃ and 500g, so that the recovery rate of the cracked product is improved under the condition of not influencing the integrity of a nuclear membrane.
4. The first filtration treatment is to remove the unlysed tissue and cell pellet while collecting as much cell suspension as possible through a cell filter having a diameter of 40 μm; the second filtration treatment and the third filtration treatment are carried out by filtering through a cell filter with the diameter of 40 μm, and the background influence on the final detection is reduced after cell debris and tissue masses are filtered.
5. Premixing PI in buffer II (LB) saves experimental time.
6. The addition of 1% FBS in buffer III (NB) helps to maintain cell viability without affecting the results of subsequent experiments.
7. Before the cell nucleus is subjected to single cell whole genome sequencing, the cell nucleus is further subjected to heavy suspension treatment, so that the concentration of the cell nucleus is 1000 nuclei/mu L, and the accuracy of a sequencing result is further increased;
8. the extraction method of the cell nucleus provided by the invention has high extraction efficiency: the integrity of the cell nucleus is good; the genome damage is small; the amplification uniformity is good. The application of the method can obtain enough mononuclear from less frozen tumor tissues for single cell whole genome sequencing.
9. The method for extracting the cell nucleus has high simplicity, only needs simple reagent consumables and does not need expensive separation equipment;
drawings
FIG. 1 is a schematic view of a photograph of nuclei of cells according to an embodiment of the present invention and a control after light-microscopic and PI staining;
FIG. 2 is a diagram showing the quality control results of the nuclear genomic DNA library construction according to an embodiment of the present invention;
FIG. 3 is a diagram showing the quality control results of the nuclear genomic DNA library construction according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of the quality control results of the A549 single cell genome DNA library construction;
FIG. 5 is a schematic representation of electron micrograph results of nuclear genomic DNA for downstream library construction, obtained according to an embodiment of the present invention and an unoptimized method.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
According to one embodiment of the invention, the invention provides a method for separating cell nucleus from frozen tumor tissue and cultured cells simply, conveniently and efficiently, the obtained mononuclear suspension is suitable for single cell whole genome amplification and downstream sequencing, and can be applied to researches such as tumor heterogeneity of single cell level on samples which are difficult to obtain fresh tissues and can be replaced by frozen tissues.
According to one embodiment of the invention, the invention is suitable for the nuclear separation of frozen tumor tissue, and the content mainly comprises 4 parts: reagent preparation, tissue cell lysis, cell nucleus cleaning and nuclear suspension detection.
(I) preparation of reagents
1. Buffer No. one (WB): 10-20mM Tris-HCl (pH 7.5-7.8), 10-20mM NaCl, 50-100mM KCl, 2-10mM MgCl2，5-15mM CaCl20.04% BSA, 1mg/mL PI and 0.2U/. mu.L RNase inhibitor;
2. buffer No. two (LB): 1mL of WB was added 2uL of NP-40.
3. Buffer No. three (NB): D-Hanks buffer containing 1% FBS, 1mM DTT.
(II) tissue cell lysis
Precooling at 1.4 ℃: PBS buffer, WB, LB and NB as described above
2. Placing a 6cm plastic culture dish on ice for precooling, adding 4mL precooled PBS (phosphate buffer solution) to wash tissues, removing culture solution from cells if the cells are cultured, washing with precooled PBS, digesting with pancreatin for 200g, centrifuging for three minutes, removing supernate, washing with precooled PBS, centrifuging and discarding the supernate for later use;
3. if the tumor tissue is detected, 1-3 mg of the tissue is placed in a pre-cooled centrifuge tube and ground to 0.2mm on ice with a pre-cooled grinding pestle3Placing the fragments on ice for 8-10 min;
4. the tissue is broken up by blowing and sucking for 10-15 times, and filtered into a flow tube by a 70-micron cell filter.
(III) Nuclear cleaning
1. Centrifuging the nuclear suspension at 4 ℃ for 5min at 500 g;
2. carefully remove the supernatant without encountering nuclear precipitates;
3. adding 0.3mL of precooled buffer solution I (WB), and gently blowing and sucking for 10 times;
4. filtering the solution into a new centrifuge tube by using a 40 mu m cell filter;
centrifuging at 5.4 deg.C for 5min at 300 g;
6. 0.2mL of pre-cooled buffer III (NB) was added, gently pipetted 10 times and resuspended and filtered through a 40 μm cell filter.
(IV) detection of nuclear suspensions
The nuclear concentration and lysis effect were measured using a Counter II FL Automated Cell Counter and a trypan blue mirror.
Example 1
(I) preparation of reagents
1.WB：10-20mM Tris-HCl(pH 7.5-7.8)，10-20mM NaCl，50-100mM KCl，2-10mM MgCl2，5-15mM CaCl20.04% BSA, 1mg/mL PI and 0.2U/. mu.L RNase inhibitor;
LB: 1mL of WB was added with 2uL of NP-40;
3. D-Hanks buffer containing 1% FBS, 1mM DTT.
(II) tissue cell lysis
1. Pre-cooling PBS and prepared WB, LB and NB;
2. placing a plastic culture dish centrifuge tube and the like on ice for precooling, and adding 4mL of precooled PBS into the culture dish to clean tissues, wherein the tissue mass is not more than 3 mg;
3. putting 3mg of tissues into a precooled centrifugal tube, grinding the tissues on ice by using a precooled grinding pestle to obtain 0.2mm3 fragments, and standing the fragments on ice for 5-10 min;
4. blowing and sucking for 10-15 times to break up the tissues, and filtering the tissues into a flow tube by using a 70-micron cell filter;
(III) Nuclear cleaning
1. Centrifuging the nuclear suspension at 4 ℃ for 5min at 500 g;
2. carefully remove the supernatant without encountering nuclear precipitates;
3. adding 0.3mL of precooled buffer solution I (WB), and gently blowing and sucking for 10 times;
4. filtering the solution into a new centrifuge tube by using a 40 mu m cell filter;
centrifuging at 5.4 deg.C for 5min at 300 g;
6. 0.2mL of pre-cooled buffer III (NB) was added, gently pipetted 10 times and resuspended and filtered through a 40 μm cell filter.
(V) detection of nuclear suspension
Mu.l of the nuclear suspension was used to determine the nuclear concentration and lysis effect using a Counter II FL Automated Cell Counter and trypan blue-light microscope. The total cell concentration was found to be 2.46 x 106mL, with viable cell concentration of 6.5 x 1043% in/mL, dead cell concentration 2.39 x 106mL, 97% indicates that a single suspended nucleus is obtained, no cell debris, 97% lysis; the total cell concentration tested by repeated sampling was 2.42 x 106mL, with a viable cell concentration of 4.69 x 1042% dead cells/mL, 2.38 x 106the/mL content was 98%, indicating that a single suspended nucleus was obtained, with no cell debris and a lysis rate of 98%. The total cell concentration was 1 x 10 as detected by PI staining5At the condition of/mL, 9.37 x 104The staining rate was 93.7% when the cells were stained.
(VI) genome amplification homogeneity detection
1000 cell nuclei are taken to extract genome DNA for MDA amplification, and amplification uniformity is detected by a fluorescent quantitative PCR method, wherein the primer sequences of eight sites of the human genome amplification uniformity experiment are shown in Table 1. The RUV value of the unamplified genomic DNA should be 1, with a RUV value close to about 1 indicating better uniformity of amplification at that site. The RUV value of at least 6 sites in a sample is between 0.25 and 4, so that the downstream library construction can be carried out. The results are shown in table 2, the eight sites on the human genome in the detection results are uniformly amplified, meet the standard and are suitable for downstream genome sequencing.
TABLE 1 primers for eight sites of the human genome amplification homogeneity experiment
TABLE 2 examples and comparative examples and the relative homogeneity values of the amplification of the single nucleus human genome obtained by the examples
(Note: the RUV value of the unamplified genomic DNA should be 1, a RUV value close to about 1 indicates that the amplification uniformity at that site is better; downstream library construction can only be performed if the RUV value of at least 6 sites in a sample is between 0.25 and 4.)
Comparative example 1 (Using a commercial cell isolation kit Cat #52009-10)
1. Prepare buffer, pre-cool at 4 ℃:
BSA was added to the LB solution before the start of the experiment to a final concentration of 1%
2. Inserting the centrifugal tube on ice for precooling, and adding 1mL of LB;
the volume is not more than 3mm3The frozen tissue samples were added to LB and ground thoroughly with a grinding pestle;
3. standing on ice for 1-10 min for full cracking;
4. filtering with 70 μm cell filter to obtain nuclear suspension; transferring the suspension into a new precooled centrifuge tube;
5. centrifuging the filtrate at 4 deg.C for 5min at 500 g;
6. the supernatant was aspirated and the precipitate was retained;
7. adding 300uL PB1, fully blowing, beating and uniformly mixing;
8. insert pipette tip to the bottom of centrifuge tube, add 600uL PB2 slowly;
9. insert pipette tip to the bottom of centrifuge tube, add 600uL PB3 slowly;
centrifuging at 10.4 deg.C and 500g for 5min to obtain LB + PB1 layer, PB2 layer and PB3 layer from top to bottom;
11. slowly absorbing the upper layer 1100uL of liquid by using a liquid transfer gun;
12. sucking the liquid at the junction of the PB2 layer and the PB3 layer of about 150uL by a P200 pipette, namely obtaining a cell nucleus layer;
13. adding 0.5mL of NB pre-cooled at 4 ℃ into the extracted cell nucleus layer, and passing through a cell filter with the diameter of 40 μm;
14. centrifuging the suspension at 4 ℃ for 5min at 500 g;
15. the nuclear pellet was resuspended in 0.2mL of pre-cooled 4 ℃ NB. The desired suspension of nuclei is obtained.
(V) detection of nuclear suspension
Mu.l of the nuclear suspension was used to determine the nuclear concentration and lysis effect using a Counter II FL Automated Cell Counter and trypan blue-light microscope. The total cell concentration was found to be 3.1 x 106mL, with a viable cell concentration of 8.45 x 10497.6% in/mL, dead cell concentration 3.02 x 106mL, 2.4%, found by PI staining at a total cell concentration of 1 x 105In the case of/mL, only 1.37 x 104the/mL is the staining of the cells, i.e., the staining rate is 13.7%, which is not in compliance with the standards for downstream amplification and sequencing.
(VI) genome amplification homogeneity detection
1. And extracting genome DNA from 1000 cell nuclei for MDA amplification, and detecting the amplification uniformity by a fluorescent quantitative PCR method. As a result, as shown in Table 2, the amplification of eight sites on the human genome was heterogeneous, and downstream genome sequencing could not be performed.
Comparative example 2 (intact Single human tumor cell)
(I) preparation of reagents
1. Pre-heating cell culture solution, PBS and pancreatin, and pre-cooling with 2% FBS-PBS;
(II) enzymatic digestion of cultured cells
1. Sucking away the cell culture solution in the culture dish, and adding 5mL of pre-warmed PBS into the culture dish to wash the cells twice;
3. absorbing PBS, adding 1mL of pre-warmed pancreatin into a culture dish, and digesting until cells are loose;
3. adding 8mL of culture solution containing serum to terminate digestion, sucking for 10-15 times, and filtering the suspension into a 15mL centrifuge tube by using a 70-micron cell filter;
(III) preparation and cleaning of single cell suspension of cultured cells
1. Centrifuging the cell suspension at normal temperature of 200g for 5 min;
2. carefully remove the supernatant without encountering cell pellet;
3. resuspending the cell centrifugation pellet with 1% FBS-containing PBS, gently pipetting 10 times (five) to detect the single cell suspension
10 μ l of the single Cell suspension was used to determine the Cell concentration and the proportion of viable cells using a Counter II FL Automated Cell Counter and trypan blue light. The total cell concentration was found to be 9.26 x 106mL, with a viable cell concentration of 8.98 x 106The cell viability is high and the cell activity is good, wherein the cell viability is 96.9 percent.
(VI) genome amplification homogeneity detection
1. Selecting 48 single cell nucleuses by CellRaft for MDA amplification, randomly selecting one cell nucleus and detecting the amplification uniformity by a fluorescent quantitative PCR method. As shown in Table 2, the eight sites on the human genome were amplified uniformly and were suitable for downstream genome sequencing.
The photographs of the cell nuclei of the examples of the present invention and the cell nuclei of the control example after the light microscope and PI staining are shown in fig. 1, and the cell nuclei (A, B) of the examples of the present invention have high integrity, can be stained, and have better quality than the comparative example (C, D).
The result of the quality test of the nuclear genome DNA library of the embodiment 1 of the invention is shown in FIG. 2, the library fragments are mainly concentrated between 350 and 550bp, the peak value is 514bp, and the standard of on-computer sequencing is met. The quality of the library downstream in example 1 of the invention is superior to that of the library constructed downstream in the method before optimization.
The result of the quality test of the nuclear genome DNA library of comparative example 1 is shown in FIG. 3, the library fragments are mainly concentrated between 350-700bp, and double peaks appear, which are 470bp and 625bp respectively, and do not meet the on-machine sequencing standard.
The result of the quality test of the A549 single cell genome DNA library construction is shown in figure 4, the library fragments are mainly concentrated between 400-550bp, the peak value is 530bp, and the standard of on-machine sequencing is met. The quality of the downstream library of the invention is better than that of the library constructed downstream by the method before optimization.
As shown in FIG. 5, the cell nucleus (Lane 1) fragments of example 1 of the present invention are more concentrated in the electrophoretogram of the library constructed from the whole genome of the cell nucleus obtained in example 1 of the present invention (Lane 1) and the cell nucleus obtained in comparative example 1(Lane 2), which indicates that the integrity of the genomic DNA is better, the quality is better than that before optimization, the product of comparative example 1, the genomic DNA is seriously degraded, and obvious diffusion can be seen, and DNA sequencing cannot be performed.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
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Claims (10)
1. A method of extracting nuclei, the method comprising the steps of:
1) performing mechanical sample cracking treatment in a buffer solution II (LB), and performing ice placement treatment and blowing treatment to obtain a cracking product;
2) performing first filtration treatment and first centrifugation treatment on the lysate to obtain a first centrifugation cell nucleus sediment;
3) performing and washing the first centrifugal cell nucleus sediment in a first buffer solution (WB) for first resuspension treatment and second filtration treatment, and then performing second centrifugation treatment to obtain a second centrifugal cell nucleus sediment;
4) performing second resuspension and uniform mixing treatment on the second centrifugal cell nucleus sediment in a No. three (NB) buffer solution, and performing third filtration treatment to obtain a cell nucleus suspension, wherein the cell nucleus suspension is further subjected to resuspension treatment before single cell whole genome sequencing and amplification uniformity detection, and the concentration of the cell nucleus is 1000 nuclei/mu L after the resuspension treatment;
wherein the sample is an ex vivo sample;
the buffer (WB) No. one comprises: 10-20mM Tris-HCl (pH 7.5-7.8), 10-20mM NaCl, 50-100mM KCl, 2-10mM MgCl2，5-15mM CaCl20.04% BSA, 1mg/mL PI andand 0.2U/. mu.L RNase inhibitor;
the mass-to-volume ratio of the sample to the buffer solution I (WB) is 5mg:0.5 mL;
the second buffer solution (LB) is the first buffer solution (WB) containing 0.2% NP-40 (mass-to-volume ratio);
the buffer solution III (NB) is a D-Hanks buffer solution containing 1% FBS and 1mM DTT;
before use, the buffer solution I (WB), the buffer solution II (LB) and the buffer solution III (NB) are pre-cooled at 4 ℃;
the volume of the sample after the crushing treatment in the step 1) is less than or equal to 0.2mm3；
The ice standing treatment time in the step 1) is 8-10min, the ice is uniformly mixed by using a liquid transfer gun, and the blow beating treatment in the step 1) is 10-15 times;
the first centrifugal treatment in the step 2) is carried out for 5min at the temperature of 4 ℃ and at the speed of 500 g;
the second filtration treatment in step 3) was performed through a cell filter having a diameter of 40 μm;
the second filtration treatment in step 4) was performed through a cell filter having a diameter of 40 μm.
2. The method for extracting nuclei of claim 1, wherein the mechanical lysis treatment is performed in step 1) by using a pestle.
3. The method for extracting nuclei of claim 1, wherein the first filtering treatment in the step 2) is performed by a cell filter having a diameter of 70 μm.
4. The method for extracting cell nucleus according to claim 1, wherein the first centrifugation treatment in step 2) is performed at 500g at 4 ℃ for 5 min.
5. The method for extracting cell nucleus according to claim 1, wherein the amount of buffer solution number one (WB) used in the first resuspension process in step 3) is 0.5 mL.
6. The method for extracting cell nucleus according to claim 1, wherein the second centrifugation of cell nucleus precipitation in step 4) is performed with the second suspension mixing treatment in 0.2mL of buffer No. three (NB).
7. A method for extracting a cell nucleus of an ex vivo frozen tumor tissue, based on the method for extracting a cell nucleus of claim 1, comprising:
step A1, pre-cooling the second buffer solution at 4 ℃, mechanically crushing the in-vitro frozen tumor tissue in the second buffer solution (LB), wherein the mass-volume ratio of the in-vitro frozen tumor tissue to the second buffer solution is not more than 5mg:0.5mL, and the volume of the in-vitro frozen tumor tissue after mechanical crushing is not more than 0.2mm3Performing ice treatment on the mechanical crushing product for 5-10 min, and uniformly mixing by using a liquid transfer gun; then, blowing and beating the ice-setting processed product for 10-15 times to obtain a blown and beaten processed product;
step A2, subjecting the blow-beating processed product obtained in step A1 to a first filtration treatment, wherein the first filtration treatment is performed through a cell filter with the diameter of 70 μm, the filtrate after the first filtration treatment forms a lysate, and the lysate is subjected to a first centrifugation treatment for 5min at 4 ℃ and 500g to obtain a cell nucleus precipitate;
step A3, resuspending the cell nucleus precipitate obtained in step A2 in the buffer solution I (WB), wherein the amount of the buffer solution I (WB) is 0.5mL, and the resuspended buffer solution I (WB) forms a first resuspended cell nucleus suspension;
step A4, subjecting the first resuspended cell nucleus suspension to a second filtration treatment, wherein the second filtration treatment is performed by a cell filter with the diameter of 40 μm, and the filtrate after the second filtration treatment constitutes a second cell nucleus filtrate;
step A5, performing second centrifugation treatment on the second cell nucleus filtrate for 5min at the temperature of 4 ℃ and the weight of 300g to obtain cell nucleus precipitates after the second centrifugation treatment;
step A6, resuspending the cell nucleus precipitate after the second centrifugal treatment by using 0.2mL of buffer solution No. three (NB) pre-cooled at 4 ℃, re-suspending and uniformly mixing, and then performing third filtration treatment to obtain a cell nucleus suspension of the in-vitro frozen tumor tissue;
before single-cell whole-gene amplification and amplification uniformity detection are carried out on the cell nuclei in the cell nucleus suspension of the in-vitro frozen tumor tissue, re-suspending treatment is carried out, and the concentration of the cell nuclei after re-suspending treatment is 1000/mu L.
8. A method of extracting nuclei of cultured adherent cells, based on the method of extracting nuclei of claim 1, comprising:
step B1, pre-cooling the second buffer solution at 4 ℃, mechanically crushing the cultured adherent cells in the second buffer solution (LB), wherein the mass-to-volume ratio of the cultured adherent cells to the second buffer solution is not more than 5mg:0.5mL, carrying out ice treatment on the product of the mechanical crushing treatment for 5-10 min, and uniformly mixing by using a liquid transfer gun; then, blowing and beating the ice-setting processed product for 10-15 times to obtain a blown and beaten processed product;
step B2, subjecting the blow-beating processed product obtained in the step A1 to a first filtration treatment, wherein the first filtration treatment is performed through a cell filter with the diameter of 70 μm, the filtrate after the first filtration treatment forms a lysate, and the lysate is subjected to a first centrifugation treatment at 4 ℃ and 500g for 5min to obtain a cell nucleus precipitate;
step B3, the cell nucleus sediment is re-suspended in the buffer solution I (WB), the amount of the buffer solution I (WB) is 0.5mL, and the re-suspended buffer solution I (WB) forms a first re-suspended cell nucleus suspension;
step B4, performing a second filtration treatment on the first resuspended cell nucleus suspension, wherein the second filtration treatment is performed through a cell filter with the diameter of 40 μm, and the filtrate after the second filtration treatment forms a second cell nucleus filtrate;
step B5, performing second centrifugation treatment on the second cell nucleus filtrate for 5min at the temperature of 4 ℃ and the weight of 300g to obtain cell nucleus precipitates after the second centrifugation treatment;
step B6, resuspending the cell nucleus precipitate after the second centrifugal treatment by using 0.2mL of No. three buffer solution (NB) precooled at 4 ℃, uniformly suspending and mixing, and then performing third filtration treatment to obtain a cell nucleus suspension of the cultured adherent cells;
wherein, before the karyon in the karyon suspension of the cultured adherent cells is subjected to single-cell whole-gene amplification and amplification uniformity detection, the karyon suspension is subjected to heavy suspension again, and the concentration of the karyon after the heavy suspension is 1000/mu L.
9. A method of single cell sequencing, based on the method of extracting nuclei of claim 1, the method steps of single cell sequencing comprising:
step C1, extracting the cell nucleus in the sample to be tested by using the method for extracting the cell nucleus of claim 1;
and C2, performing single cell whole genome sequencing on the cell nucleus so as to obtain a sequencing result.
10. The method of claim 7, wherein the nuclei are subjected to single cell whole genome sequencing, and then are used for the research of tumor evolution mechanism, cancer precise typing, tumor drug resistance mechanism and efficacy prediction.
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