CN112280829A - Kit, sample marking method and single cell sequencing method - Google Patents
Kit, sample marking method and single cell sequencing method Download PDFInfo
- Publication number
- CN112280829A CN112280829A CN202011200481.2A CN202011200481A CN112280829A CN 112280829 A CN112280829 A CN 112280829A CN 202011200481 A CN202011200481 A CN 202011200481A CN 112280829 A CN112280829 A CN 112280829A
- Authority
- CN
- China
- Prior art keywords
- sample
- sequence
- cell
- sequencing
- biotin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 74
- 238000000034 method Methods 0.000 title claims description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 175
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 116
- 229960002685 biotin Drugs 0.000 claims abstract description 59
- 239000011616 biotin Substances 0.000 claims abstract description 59
- 235000020958 biotin Nutrition 0.000 claims abstract description 58
- 239000002773 nucleotide Substances 0.000 claims abstract description 34
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 34
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 30
- 238000002372 labelling Methods 0.000 claims abstract description 30
- 108010062580 Concanavalin A Proteins 0.000 claims abstract description 28
- 108090001008 Avidin Proteins 0.000 claims abstract description 24
- 108020001507 fusion proteins Proteins 0.000 claims description 21
- 102000037865 fusion proteins Human genes 0.000 claims description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims description 18
- 108010090804 Streptavidin Proteins 0.000 claims description 14
- 108020004999 messenger RNA Proteins 0.000 claims description 10
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 claims description 9
- 102100035304 Lymphotactin Human genes 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 210000000170 cell membrane Anatomy 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- 239000012528 membrane Substances 0.000 abstract description 4
- 239000006285 cell suspension Substances 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 150000007523 nucleic acids Chemical class 0.000 description 17
- 210000004940 nucleus Anatomy 0.000 description 14
- 239000008363 phosphate buffer Substances 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000005406 washing Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000013461 design Methods 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 210000000633 nuclear envelope Anatomy 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000012864 cross contamination Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108010038196 saccharide-binding proteins Proteins 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 108010075254 C-Peptide Proteins 0.000 description 1
- 241000220451 Canavalia Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 102000023852 carbohydrate binding proteins Human genes 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The technical scheme of the invention is that a kit is designed, the kit comprises a sample label sequence with a biotin label, the sample label sequence comprises a bar code nucleotide sequence for specifically labeling a sample, the kit also comprises a concanavalin A or an analogue thereof with the biotin label, and avidin for connecting the sample label sequence and the concanavalin A or the analogue thereof, before the mixed sample of single cell sequencing, the sample is pretreated by using a kit, so that the concanavalin A or the analogue thereof connected with the sample label sequence is connected on a cell membrane or a cell nucleus membrane, so that the cells of each sample carry different sample label sequences, thereby being capable of clearly distinguishing which sample the cells come from in the subsequent single cell sequencing result, the scheme is simple to operate, low in cost and good in effect, can be universally used for marking cells and cell nucleuses, and reduces the cost of single cell sequencing.
Description
The application requires application number 202010268124.3, applied on 04/07/2020, entitled priority of Chinese patent application of "a kit, a sample labeling method, and a single cell sequencing method".
Technical Field
The invention relates to the technical field of biology, in particular to a kit, a sample marking method and a single cell sequencing method.
Background
The single cell sequencing technology greatly advances the field of genomics, and enables different cell types to be finely distinguished by revealing unique subtle changes of each cell, so that scientists can possibly research molecular mechanisms at the single cell level.
With the development of technology, in a single cell sequencing experiment, 1-10 ten thousand single cells can be captured simultaneously, and due to the high cost of the single cell sequencing experiment, if the sequencing cost of each cell is reduced, the throughput of single cell sequencing can only be increased. However, for some single cell sequencing experiments, a small number of cells can meet the requirement of the experiment or the number of cells of a sequencing sample is not large, and in order to reduce the cost, it is now common practice to mix different single cell sequencing samples together to perform a high-throughput single cell sequencing experiment.
In order to accurately distinguish cells from different single cell sequencing samples in subsequent analysis, the cells must be labeled before mixing the different single cell sequencing samples. There are two main methods of labeling currently in use: first, by expressing a specific nucleic acid tag in a cell; secondly, cells are labeled with antibodies carrying specific nucleic acid tags.
The marking methods currently used mainly have the following disadvantages: the first method requires a long pretreatment of the cells, is complicated to operate, and cannot be used for freshly isolated single cells from tissues. The second method requires prior knowledge of the antigen expression on the surface of different cells, and cannot be used if the two cells are not very different; in addition, such tags are expensive to produce and cannot be used for nuclear labeling.
Disclosure of Invention
The invention mainly aims to provide a kit, and the method for marking the single cell sequencing sample by the kit has the advantages of simple operation, low cost and good effect, and can be universally used for marking cells and cell nucleuses.
In order to achieve the above object, in a first aspect, the present invention provides a kit comprising:
a first reagent comprising a sample tag sequence comprising a barcode nucleotide sequence for specifically labeling a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labeled at either end with biotin or an analog thereof;
a second agent comprising concanavalin A or an analog thereof labeled with biotin or an analog thereof;
and the third reagent comprises avidin or an analogue thereof, and the avidin or the analogue thereof is used for connecting the biotin or the analogue thereof in the first reagent and the biotin or the analogue thereof in the second reagent.
In a second aspect, the present invention also provides another kit comprising:
a sample tag sequence comprising a barcode nucleotide sequence that specifically labels a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labelled at either end with biotin or an analogue thereof;
a fusion protein of concanavalin a or an analog thereof and avidin or an analog thereof, for linking to biotin or an analog thereof in the sample tag sequence.
In a third aspect, the present invention provides a further kit, comprising:
a sample tag sequence comprising a barcode nucleotide sequence specifically labeling a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labeled at either end with biotin or an analog thereof, the sample tag sequence being linked via avidin or an analog thereof to concanavalin a or an analog thereof labeled with biotin or an analog thereof.
Optionally, the other end of the barcode nucleotide sequence is linked to a first primer binding sequence.
Optionally, the cell tag binding sequence comprises a second primer binding sequence or a poly A tail sequence.
Optionally, the avidin is streptavidin.
In a fourth aspect, the present invention further provides a sample labeling method, comprising,
resuspending the cell sample or the cell nucleus sample in a buffer solution to obtain a sample to be marked;
and adding the reagent in the kit of the third aspect into the sample to be marked for incubation to obtain the marked sample.
In a fifth aspect, the present invention also provides a single cell sequencing method, wherein a single cell sequenced sample is pretreated by using the kit of any one of the first to third aspects.
Optionally, the single cell sequencing is single cell mRNA sequencing.
Optionally, the single cell sequencing is single cell ATAC sequencing.
The technical scheme of the invention is that a kit is designed, the kit comprises a sample label sequence with a biotin label, the sample label sequence comprises a bar code nucleotide sequence for specifically labeling a sample, the kit also comprises a concanavalin A or an analogue thereof with the biotin label, and avidin for connecting the sample label sequence and the concanavalin A or the analogue thereof, before the mixed sample of single cell sequencing, the sample is pretreated by using a kit, so that the concanavalin A or the analogue thereof connected with the sample label sequence is connected on a cell membrane or a cell nucleus membrane, so that the cells of each sample carry different sample label sequences, thereby being capable of clearly distinguishing which sample the cells come from in the subsequent single cell sequencing result, the scheme is simple to operate, low in cost and good in effect, can be universally used for marking cells and cell nucleuses, and reduces the cost of single cell sequencing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a sample tag sequence according to an embodiment of the present invention;
FIG. 2 is a sample tag sequence according to another embodiment of the present invention;
FIG. 3 is a schematic diagram of the structure of a complex binding membrane for labeling a sample according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of experiments in which HEK293 cells were labeled with complexes of a labeled sample according to an embodiment of the present invention;
FIG. 5 is a graph showing the sequencing results of single-cell mRNA after labeling cells with complexes of a sample according to an embodiment of the present invention;
FIG. 6 is a graph showing the results of experiments in which the complexes of the labeled samples labeled with the present invention labeled with MEF nuclei;
FIG. 7 is a graph showing the sequencing results of single cell ATAC after labeling the cell nucleus with the complex of the labeled sample according to the embodiment of the present invention;
FIG. 8 is a graph showing the results of purification and expression of monomeric streptavidin-concanavalin A fusion protein according to an embodiment of the present invention;
FIG. 9 is a graph showing the results of an experiment in which monomeric streptavidin-concanavalin A fusion protein labeled cells according to an embodiment of the present invention are obtained;
FIG. 10 is a graph showing the results of an experiment for detecting complex cross-contamination of a labeled sample according to an embodiment of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a kit, which comprises a reagent I, wherein the reagent I comprises a sample label sequence, the sample label sequence comprises a bar code nucleotide sequence for specifically labeling a sample, either end of the bar code nucleotide sequence is connected with a cell label binding sequence, and either end of the sample label sequence is labeled with biotin or analogues thereof; a second agent comprising concanavalin A or an analog thereof labeled with biotin or an analog thereof; and the third reagent comprises avidin or an analogue thereof, and the avidin or the analogue thereof is used for connecting the biotin or the analogue thereof in the first reagent and the biotin or the analogue thereof in the second reagent.
According to the technical scheme, the cells of each sample are provided with different sample label sequences before the mixed samples are subjected to single cell sequencing, so that which sample the cells come from can be clearly distinguished in the subsequent single cell sequencing result.
Specifically, referring to fig. 1 and fig. 2, the sample tag sequence is substantially a nucleic acid tag, each position of the barcode nucleotide sequence may be A, T, C, G bases, and different barcode nucleotide sequences are obtained by random arrangement of the bases, the barcode nucleotide sequence is a characteristic sequence of the sample tag sequence, the length of the characteristic sequence can be designed at will, and is preferably 8-10 bases in length, and too short a barcode nucleotide sequence may cause insufficient tag types and too long a barcode nucleotide sequence may cause unnecessary waste.
Specifically, the cell tag binding sequence is used for being connected with a cell tag in single cell sequencing subsequently, the cell tag is respectively connected with a sequencing substance in a cell and a sample tag sequence and is respectively sequenced, and finally, which sample the sequencing substance comes from is known according to the sequencing substance with the same cell tag and the sample tag sequence. The cell tag sequence may be attached to the 5 'end or 3' end of the barcode nucleotide sequence.
Optionally, the sample tag sequence further comprises a first primer binding sequence for subsequent PCR amplification of the sample tag sequence, the length of the first primer binding sequence is typically between 16-28 bases, and the first primer binding sequence design is required to follow the primer design principle. Accordingly, the first primer binding sequence is attached to the barcode nucleotide sequence at the end not attached to the cell tag sequence.
Alternatively, referring to fig. 1, the cell tag binding sequence is a second primer binding sequence, which also needs to follow the principle of primer design, and the second primer binding sequence is used for subsequent connection with a cell tag in single cell sequencing through base complementary pairing, during the single cell sequencing process, the cell tag is respectively connected with a sequencing material in a cell and a sample tag sequence, and is respectively sequenced, and finally, which sample the sequencing material comes from is known according to the sequencing material and the sample tag sequence with the same cell tag.
Furthermore, random sequences for increasing the length of the sample tag sequence can be included between the first primer binding sequence and the barcode nucleotide sequence and between the second primer binding sequence and the barcode nucleotide sequence, so that the recovery efficiency of the sample tag sequence in the subsequent single cell sequencing can be increased.
Alternatively, referring to fig. 2, the cell tag binding sequence is a poly-A sequence tail, the sample tag sequence is dedicated to mRNA sequencing, and the single-cell mRNA sequencing has a poly-T tail sequence, so that the sample tag sequence can bind with the cell tag sequence, thereby sequencing the sequencing material with the cell tag and the sample tag sequence with the cell tag respectively, and knowing from which sample the sequencing material comes from according to the same cell tag.
Specifically, each avidin molecule consists of 4 subunits, and can be closely bound to 4 biotin molecules. Preferably, the avidin is streptavidin, and the binding specificity of streptavidin to biotin is higher. Referring to FIG. 3, streptavidin is a tetrameric protein, 66kDa in size. One streptavidin can be highly specific to four biotin molecules, with very strong affinity between the two, and the dissociation constant of the streptavidin-biotin complex is at 10-14In the order of mol/L.
Canavarin A is a carbohydrate-binding protein, tetrameric globulin, with molecular weight of 102000, 237 amino acid residues per subunit, molecular weight of 25500, and a carbohydrate-binding site. Since the surface of the cell membrane or the nuclear membrane is provided with a large amount of glycoprotein, the concanavalin A can be combined with the glycoprotein on the cell membrane or the nuclear membrane, so that the nucleic acid label, namely the sample marker sequence is marked on the surface of the cell membrane or the nuclear membrane. It will be appreciated that the complex is not limited to the use of concanavalin A and that other functional proteins having similar functions to concanavalin A may be used.
Preferably, the molar ratio of the streptavidin to the sample tag sequence, the biotin-labeled concanavalin a, or the analog thereof is 1: 1: 1.
since the biotin-bearing nucleic acid tag and biotin-bearing concanavalin a compete with each other in binding to streptavidin, 1: 1: the ratio of 1 is the most stable ratio of the compound. It is understood that the molecular ratio of streptavidin to the biotin-labeled nucleic acid tag and the biotin-labeled concanavalin A may be in other ratios as long as the function of the complex is not affected.
Specifically, when the kit is used, avidin and biotin-labeled concanavalin a are dissolved in 50% glycerol, the biotin-labeled sample tag sequence is dissolved in double distilled water, appropriate concentrations are respectively prepared, avidin and the biotin-labeled sample tag sequence are incubated under the incubation condition of room temperature for 5 minutes, then biotin-labeled concanavalin a is added for incubation, a labeled sample complex shown in fig. 3 is obtained, and the complex is incubated with a sample, so that a cell or cell nucleus sample has the sample tag sequence.
In a second aspect, the present invention also provides a kit comprising:
a sample tag sequence comprising a barcode nucleotide sequence that specifically labels a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labelled at either end with biotin or an analogue thereof;
a fusion protein of concanavalin a or an analog thereof and avidin or an analog thereof, for linking to biotin or an analog thereof in the sample tag sequence.
Specifically, unlike the first embodiment, the kit of this embodiment performs fusion expression of concanavalin a or its analog and avidin to obtain a fusion protein having a membrane-binding function of concanavalin a and a biotin-binding function of avidin, so that the fusion protein can be linked to a biotin-labeled sample tag sequence without biotin labeling.
Specifically, in use, the fusion protein is incubated with a sample tag sequence to obtain a complex similar to the labeled sample shown in FIG. 3, and the complex is incubated with a cell or cell nucleus sample to label the sample.
In a third aspect, the present invention provides a further kit, comprising:
a sample tag sequence comprising a barcode nucleotide sequence specifically labeling a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labeled at either end with biotin or an analog thereof, the sample tag sequence being linked via avidin or an analog thereof to concanavalin a or an analog thereof labeled with biotin or an analog thereof.
Specifically, unlike the first and second embodiments, the reagent in the kit of this embodiment is a complex, and the preparation method of the complex includes incubating avidin with a biotin-labeled sample tag sequence to obtain a first mixture, and adding biotin-labeled concanavalin a or an analog thereof to the first mixture to incubate to obtain a complex labeling the sample.
Specifically, avidin and biotin-labeled concanavalin a are dissolved in 50% glycerol, and biotin-labeled sample tag sequences are dissolved in double distilled water, and are respectively prepared at appropriate concentrations, and then incubated according to the method, wherein the incubation condition can be room temperature incubation for 5 minutes.
In a fourth aspect, the present invention further provides a sample labeling method, comprising,
resuspending the cell sample or the cell nucleus sample in a buffer solution to obtain a sample to be marked;
and adding the reagent in the kit of the third aspect into the sample to be marked for incubation to obtain the marked sample.
Specifically, if the cell nucleus sample needs to be subjected to cell nucleus extraction first, the cell nucleus extraction method is performed according to the prior art. The buffer may be a phosphate buffer or a buffer of the nucleus. And (3) resuspending the sample to obtain a single cell suspension or a single cell nuclear suspension so as to conveniently mark a sample label sequence on a cell membrane or a nuclear membrane.
Alternatively, the incubation condition is ice for 10 minutes.
In a fifth aspect, the present invention also provides a single cell sequencing method, wherein a single cell sequencing sample is pretreated by using the kit of any one of the first to third aspects, and the specific use method is described with reference to the above first to third aspects.
Specifically, after the kit of the embodiments of the first aspect to the third aspect marks the cell or the cell nucleus, the cell or the cell nucleus is provided with the sample tag sequence, so that the cell or the cell nucleus of which the sequencing result comes from can be clearly distinguished in the subsequent single cell sequencing.
Alternatively, the sample tag sequence is used for single cell mRNA sequencing when the 3' end of the sample tag sequence is designed as a poly-a tail. At this time, the cell tag sequence for single cell sequencing is poly-T tail, so the cell tag sequence can be respectively combined with the sample tag sequence and mRNA, so that amplification is carried out according to the primer combination sequence, and sequencing is respectively carried out.
Optionally, when the sample is a nucleus, the single cell sequencing is single cell ATAC sequencing. ATAC sequencing is to sequence open regions of chromatin in nuclei, and the design of the tag sequence of the sample is shown in FIG. 1 and will not be described herein.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 design of sample tag sequences
Respectively designing sample tag sequences for mRNA single cell sequencing and cell nucleus ATAC sequencing, wherein the results are that SIQ ID NO.1 and SIQ ID NO.2 are respectively provided with biotin labels at the 5' ends, the barcode nucleotide sequence is composed of 8 bases and is randomly arranged during synthesis, and the 34 th to 56 th bases and the 65 th to 175 th bases of the sample tag sequence of the ATAC sequencing are random sequences for increasing the length of the sample tag sequence.
EXAMPLE 2 preparation of complexes of labeled samples
1. Three components of the complex to make the labeled sample are prepared: biotinylated Canavalia protein A was purchased from Sigma-Aldrich, cat # C2272; streptavidin was purchased from beijing, cool ladyb technologies ltd, cat # CS 10471; the biotin-labeled sample tag nucleotide sequence was synthesized by Biotechnology engineering (Shanghai) Inc.
2. Biotin-labeled concanavalin a and streptavidin were dissolved at a concentration of 1.6uM in 50% glycerol, and the biotin-labeled sample tag nucleotide sequence was dissolved at a concentration of 0.1uM in double distilled water.
3. Preparing a molecular label: first, the streptavidin molecules and the biotin-labeled sample tag nucleotide sequences were labeled with a 1: 1, mixing, uniformly mixing, and standing at room temperature for 5 minutes. Then adding biotin-labeled canavalin A to ensure that the molar ratio of the canavalin A to the streptavidin is 1: 1, mixing, and standing at room temperature for 5 minutes.
Example 3 labeling of complexes of samples to label cells
1. Preparing cells: HEK293 cells were suspended in 0.5mL of phosphate buffer as single cells to obtain single cell suspensions, and a total of 6 single cell suspensions were set up.
2. Labeling cells: the prepared molecular tags 0. mu.l, 1.25. mu.l, 2.5. mu.l, 5. mu.l, 10. mu.l and 20. mu.l were added to the 6 single cell suspensions prepared in 1, respectively, and the mixtures were mixed and left on ice for 10 minutes.
3. Washing off the excess molecular tags: after washing the cells twice with 1mL of phosphate buffer, the cells were resuspended in phosphate buffer.
4. Measuring the number of bound molecular tags on cells: first, a standard curve of a biotin-labeled nucleic acid tag is plotted using fluorescent quantitative PCR. And (3) directly placing the marked cells into a fluorescent quantitative PCR reaction system (each reaction of 2000 cells) for measurement, and finally calculating the number of molecular labels on each cell according to a standard curve.
The results are shown in fig. 4, the number of molecular tags on each cell in each group of single cell suspension on average increases with the increase of the amount of the molecular tags, and the number of the molecular tags can reach as many as 5 ten thousand, which proves that the molecular tags of the compound obtained in example 2 have good labeling effect on the cells.
Example 4 Single cell mRNA sequencing of labelled cells
1. Preparing cells: resuspend different kinds of cells in 0.5mL phosphate buffer solution as single cells to obtain single cell suspension, and set up 20 single cell suspensions in total.
2. Labeling cells: to 20 single cell suspensions prepared in 1, 2.5 μ l of prepared 20 sample tags with different sequences were added, mixed, and left on ice for 10 minutes.
3. Washing off excess sample label: after washing the cells twice with 1mL of phosphate buffer, the cells were resuspended in phosphate buffer.
4. Measurement of the species bound to the sample tag on the cells: the single cell mRNA sequencing kit was used to capture the sample tags on the cells and the species of sample tags bound on each cell was analyzed using next generation sequencing.
As shown in FIG. 5, the molecular tags bound to each single-cell sample group were detected by second-generation sequencing, and cells containing more than 1 molecular tag were considered to be double-coated.
Example 5 labeling complexes of samples labeling nuclei
1. Preparing cell nuclei: the nucleus of MEF cell is suspended in 0.5mL of nucleus extracting solution in a single nucleus mode to obtain single nucleus suspension, and 6 groups of single nucleus suspension are arranged in total.
2. Marking cell nucleus: the prepared molecular tags 0. mu.l, 1.25. mu.l, 2.5. mu.l, 5. mu.l, 10. mu.l and 20. mu.l were added to the 6 sets of single cell nuclei suspensions prepared in 1, respectively, and the mixtures were mixed and left on ice for 10 minutes.
3. Washing off the excess molecular tags: after washing the cells twice with 1mL of the cell nucleus wash buffer, the cells were resuspended in the cell nucleus wash buffer.
4. Measurement of the number of bound molecular tags on the nucleus: first, a standard curve of a biotin-labeled nucleic acid tag is plotted using fluorescent quantitative PCR. And (3) directly placing the marked cell nucleus into a fluorescence quantitative PCR reaction system (each reaction of 2000 cell nuclei) for measurement, and finally calculating the number of the molecular tags on each cell nucleus according to a standard curve.
As shown in FIG. 6, the number of molecular tags per cell nucleus on average in each single cell nuclear suspension group increases with the increase of the amount of the molecular tags, and the number of the molecular tags per cell nucleus on average can reach as much as 12 ten thousand, which proves that the molecular tags of the complex obtained in example 2 have good labeling effect on the cell nucleus.
Example 6 Single cell ATAC sequencing of labelled nuclei
1. Preparing cells: resuspend different kinds of cells in 0.5mL phosphate buffer solution in the form of single cell nucleus to obtain single cell suspension, and set up 8 groups of single cell suspensions in total.
2. Labeling cells: and (3) adding 2.5 mu l of the prepared 8 groups of single cell suspensions with different sequences into the prepared 8 groups of single cell suspensions in the step (1), uniformly mixing, and standing on ice for 10 minutes.
3. Washing off excess sample label: after washing the cells twice with 1mL of phosphate buffer, the cells were resuspended in phosphate buffer.
4. Measurement of the species bound to the sample tag on the cells: the single cell ATAC sequencing kit was used to capture the sample tags on the nuclei and the species of the bound sample tags on each nucleus was analyzed using next generation sequencing.
As shown in FIG. 7, the molecular tags bound to each single-cell sample group were detected by second-generation sequencing, and cells containing more than 1 molecular tag were considered to be double-coated.
Example 7 preparation of "monomeric streptavidin-Canavanin A" fusion protein
1. Preparing a prokaryotic expression vector: the protein coding sequence of single-type streptavidin and canavalin A is inserted into the multiple cloning site of pGEX6P1 prokaryotic expression vector, the two sequences are separated by three GGGGS connecting peptides, and the two sequences and the upstream GST protein coding sequence are in the same coding frame.
2. Prokaryotic expression: the expression vector is transferred into escherichia coli cells and cultured, and the expression of the fusion protein is induced by IPTG.
3. Purification of the fusion protein: coli expressing the fusion protein was disrupted and the fusion protein was purified using a GST tag protein purification column. Thereafter, the eluted proteins were dialyzed and concentrated. Finally, the fusion protein was identified by SDS-page and Coomassie Brilliant blue, and the result is shown in FIG. 8, where the size of the fusion protein with GST tag was around 70kDa, which was in line with the expected size.
Example 8 labelling of cells with monomeric streptavidin-concanavalin A fusion protein
1. The fusion protein was dissolved in 50% glycerol at a concentration of 400nM, and the biotin-labeled sample tag nucleotide sequence was dissolved in double distilled water at a concentration of 0.1. mu.M.
2. Preparing cells: HEK293 cells were suspended in 0.2mL of phosphate buffer as single cells to obtain single cell suspensions, and a total of 7 single cell suspensions were set up.
3. Labeling of cells with fusion protein: 2.5. mu.l of the prepared fusion protein was added to each of the 7 single cell suspensions prepared in 2, and the resulting mixture was mixed well and left on ice for 10 minutes.
4. Excess fusion protein was washed away: after washing the cells once with 1mL of phosphate buffer, the cells were resuspended in phosphate buffer.
5. Labeling cells with nucleic acid tags: the prepared biotin-labeled nucleic acid tags 0. mu.l, 0.625. mu.l, 1.25. mu.l, 2.5. mu.l, 5. mu.l, 10. mu.l and 20. mu.l were added to the 7 single cell suspensions prepared in 4, respectively, and the mixtures were mixed and left on ice for 10 minutes.
6. Washing off excess nucleic acid tags: after washing the cells once with 1mL of phosphate buffer, the cells were resuspended in phosphate buffer.
7. Measuring the number of bound molecular tags on cells: first, a standard curve of a biotin-labeled nucleic acid tag is plotted using fluorescent quantitative PCR. And (3) directly placing the marked cells into a fluorescent quantitative PCR reaction system (each reaction of 200 cells) for measurement, and finally calculating the number of molecular labels on each cell according to a standard curve.
The results are shown in fig. 9, the number of the nucleic acid tags on each cell in each group of single cell suspension increases with the increase of the amount of the nucleic acid tags, and the number of the nucleic acid tags can reach as many as 5 ten thousand, which proves that the fusion protein obtained in example 7 has good labeling effect on the cells.
Example 9 Cross-contamination assay of sample tag sequences
To detect the possibility of cross-contamination of the sample labeled with the tag sequence, labeled and unlabeled cells are mixed and then separated to detect the number of nucleic acid tags on the cells, respectively. As shown in FIG. 10, the nucleic acid tag did not detach from the labeled cells and contaminate the unlabeled cells, demonstrating that the stability of the sample tag sequence is good.
The above description is only an alternative embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> southern university of science and technology
<120> kit, sample marking method and single cell sequencing method
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 59
<212> DNA
<213> Artificial Synthesis
<220>
<221> misc_feature
<222> (21)..(28)
<223> n is a,c,g or t
<400> 1
gctgcgctcg atgcaaaata nnnnnnnnaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59
<210> 2
<211> 209
<212> DNA
<213> Artificial Synthesis
<220>
<221> misc_feature
<222> (57)..(64)
<223> n is a,c,g or t
<400> 2
tcgtcggcag cgtcagatgt gtataagaga cagcttgtgg aaaggacgaa acaccgnnnn 60
nnnngtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 120
aagtggcacc gagtcggtgc ttttttaagc ttggcgtaac tagatcttga gacacctgtc 180
tcttatacac atctccgagc ccacgagac 209
Claims (10)
1. A kit, comprising:
a first reagent comprising a sample tag sequence comprising a barcode nucleotide sequence for specifically labeling a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labeled at either end with biotin or an analog thereof;
a second agent comprising concanavalin A or an analog thereof labeled with biotin or an analog thereof;
and the third reagent comprises avidin or an analogue thereof, and the avidin or the analogue thereof is used for connecting the biotin or the analogue thereof in the first reagent and the biotin or the analogue thereof in the second reagent.
2. A kit, comprising:
a sample tag sequence comprising a barcode nucleotide sequence that specifically labels a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labelled at either end with biotin or an analogue thereof;
a fusion protein of concanavalin a or an analog thereof and avidin or an analog thereof, for linking to biotin or an analog thereof in the sample tag sequence.
3. A kit, comprising:
a sample tag sequence comprising a barcode nucleotide sequence specifically labeling a sample, the barcode nucleotide sequence being linked at either end to a cell tag binding sequence, the sample tag sequence being labeled at either end with biotin or an analog thereof, the sample tag sequence being linked via avidin or an analog thereof to concanavalin a or an analog thereof labeled with biotin or an analog thereof.
4. A kit as claimed in any one of claims 1 to 3, wherein the barcode nucleotide sequence is linked at the other end to a first primer binding sequence.
5. A kit according to claim 4, wherein the cell tag binding sequence comprises a second primer binding sequence or a poly A tail sequence.
6. A kit according to claim 5, wherein the avidin is streptavidin.
7. A method for marking a sample, comprising,
resuspending the cell sample or the cell nucleus sample in a buffer solution to obtain a sample to be marked;
adding the reagent in the kit according to claim 3 into the sample to be marked for incubation to obtain the marked sample.
8. A method for sequencing single cells, wherein a sample for sequencing single cells is pretreated with the kit of any one of claims 1 to 6.
9. The single cell sequencing method of claim 8, wherein said single cell sequencing is single cell mRNA sequencing.
10. The single cell sequencing method of claim 8, wherein said single cell sequencing is single cell ATAC sequencing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2021/085715 WO2021204124A1 (en) | 2020-04-07 | 2021-04-06 | Sample tag sequence, kit, sample labeling method, single-cell sequencing method |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020102681243 | 2020-04-07 | ||
| CN202010268124 | 2020-04-07 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112280829A true CN112280829A (en) | 2021-01-29 |
| CN112280829B CN112280829B (en) | 2023-12-26 |
Family
ID=74353114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011200481.2A Active CN112280829B (en) | 2020-04-07 | 2020-10-30 | Kit, sample marking method and single-cell sequencing method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112280829B (en) |
| WO (1) | WO2021204124A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021204124A1 (en) * | 2020-04-07 | 2021-10-14 | 南方科技大学 | Sample tag sequence, kit, sample labeling method, single-cell sequencing method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575228A (en) * | 2009-04-08 | 2012-07-11 | 加利福尼亚大学董事会 | DNA-cell conjugates |
| US9957554B1 (en) * | 2013-12-19 | 2018-05-01 | National Technology & Engineering Solutions Of Sandia, Llc | Microfluidic platform for multiplexed detection in single cells and methods thereof |
| CN110199019A (en) * | 2016-05-02 | 2019-09-03 | Encodia有限公司 | It is analyzed using the macromolecular of nucleic acid encode |
| US20190316182A1 (en) * | 2016-12-23 | 2019-10-17 | Cs Genetics Limited | Reagents and methods for molecular barcoding of nucleic acids of single cells |
| US20200040379A1 (en) * | 2018-08-03 | 2020-02-06 | Cellular Research, Inc. | Nuclei barcoding and capture in single cells |
| CN110785488A (en) * | 2017-04-21 | 2020-02-11 | 圣拉斐尔医院有限责任公司 | Gene therapy |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280829B (en) * | 2020-04-07 | 2023-12-26 | 南方科技大学 | Kit, sample marking method and single-cell sequencing method |
-
2020
- 2020-10-30 CN CN202011200481.2A patent/CN112280829B/en active Active
-
2021
- 2021-04-06 WO PCT/CN2021/085715 patent/WO2021204124A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575228A (en) * | 2009-04-08 | 2012-07-11 | 加利福尼亚大学董事会 | DNA-cell conjugates |
| US9957554B1 (en) * | 2013-12-19 | 2018-05-01 | National Technology & Engineering Solutions Of Sandia, Llc | Microfluidic platform for multiplexed detection in single cells and methods thereof |
| CN110199019A (en) * | 2016-05-02 | 2019-09-03 | Encodia有限公司 | It is analyzed using the macromolecular of nucleic acid encode |
| US20190316182A1 (en) * | 2016-12-23 | 2019-10-17 | Cs Genetics Limited | Reagents and methods for molecular barcoding of nucleic acids of single cells |
| CN110785488A (en) * | 2017-04-21 | 2020-02-11 | 圣拉斐尔医院有限责任公司 | Gene therapy |
| US20200040379A1 (en) * | 2018-08-03 | 2020-02-06 | Cellular Research, Inc. | Nuclei barcoding and capture in single cells |
Non-Patent Citations (3)
| Title |
|---|
| LIANG FANG等: "A concanavalin A-based sample barcoding strategy for single-cell sequencing", BIORXIV, pages 13 - 15 * |
| MARLON STOECKIUSS等: "Large-scale simultaneous measurement of epitopes and transcriptomes in single cells", NAT METHODS., vol. 14, no. 9, pages 5 - 6 * |
| 刘畅等: "适配体生物传感器在生物分子及细胞检测中的应用研究", 检验医学, no. 1, pages 70 - 74 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021204124A1 (en) * | 2020-04-07 | 2021-10-14 | 南方科技大学 | Sample tag sequence, kit, sample labeling method, single-cell sequencing method |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021204124A1 (en) | 2021-10-14 |
| CN112280829B (en) | 2023-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3008094B1 (en) | Bis-biotinylation tags | |
| CN110129415B (en) | NGS library-building molecular joint and preparation method and application thereof | |
| ATE377091T1 (en) | METHOD FOR DETECTING NUCLEIC ACIDS | |
| CN101148667A (en) | Preparation and use for affinity human albumin nucleic acid aptamer | |
| CN108660191A (en) | A kind of digitlization multiple nucleic acid detection method based on coding microball reactor | |
| WO2022213656A1 (en) | Functionalized magnetic beads and one-step macromolecule capture method using bio-orthogonal chemistry of said magnetic beads | |
| CN112280829B (en) | Kit, sample marking method and single-cell sequencing method | |
| CN111500583B (en) | Aptamer for specifically recognizing bovine pregnancy-associated glycoprotein 4 and application thereof | |
| Blackwell | [41] Selection of protein binding sites from random nucleic acid sequences | |
| CN112266916B (en) | Sheep pregnancy related glycoprotein nucleic acid aptamer and application thereof | |
| CN114457083A (en) | Single-stranded DNA (deoxyribonucleic acid) aptamer group for specifically recognizing malachite green and application thereof | |
| CN113462693A (en) | Application of ssDNA aptamer in identification of iridovirus infected cells of micropterus salmoides | |
| CN112500850B (en) | Three-functional magnetic aptamer trinucleotide nanoprobe | |
| US20150050644A1 (en) | Method for Detecting Pyrophosphoric Acid Using Support Having Boronic Acid Group Immobilized Thereon | |
| CN111139285A (en) | Method for detecting contents of different target proteins in multiple samples to be detected in high-throughput manner and special kit thereof | |
| CN114540359B (en) | Aptamer capable of specifically recognizing trimethoprim, and screening and application thereof | |
| CN111548412A (en) | Hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence | |
| CN113481203B (en) | Aptamer specifically combined with human FXYD2 gamma a, derivative thereof, screening method and application | |
| CN112961859B (en) | Aptamer for specifically recognizing amantadine and application thereof | |
| CN112067602B (en) | ATP chemiluminescence detection method based on enzyme digestion-assisted unmarked aptamer sensor | |
| CN113278621B (en) | ssDNA aptamer and application thereof in identification and detection of vibrio harveyi | |
| CN111876423B (en) | Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative | |
| KR101069589B1 (en) | Single-stranded DNA aptamers specifically binding to CEA | |
| WO2022188054A1 (en) | Methods and reagents for sample multiplexing for high throughput single-cell rna sequencing | |
| CN116773805A (en) | Kit and method for detecting cell damage degree |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |