CN111378728A - Magnetic bead with molecular label primer sequence and preparation method thereof - Google Patents
Magnetic bead with molecular label primer sequence and preparation method thereof Download PDFInfo
- Publication number
- CN111378728A CN111378728A CN201811653760.7A CN201811653760A CN111378728A CN 111378728 A CN111378728 A CN 111378728A CN 201811653760 A CN201811653760 A CN 201811653760A CN 111378728 A CN111378728 A CN 111378728A
- Authority
- CN
- China
- Prior art keywords
- magnetic
- sequences
- differential
- sequence
- magnetic beads
- 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.)
- Pending
Links
- 239000011324 bead Substances 0.000 title claims abstract description 106
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 238000012163 sequencing technique Methods 0.000 claims abstract description 15
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims description 35
- 238000002156 mixing Methods 0.000 claims description 14
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 102000012410 DNA Ligases Human genes 0.000 claims description 6
- 108010061982 DNA Ligases Proteins 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000001308 synthesis method Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000005251 capillar electrophoresis Methods 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000011325 microbead Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 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
- C12Q1/6869—Methods for sequencing
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a magnetic bead with a molecular label primer sequence, which is connected with streptavidin through surface modification, and is connected with a DNA barcode sequence on the surface of the magnetic bead. The preparation method comprises the following steps: s1, synthetic DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences; s2, connecting magnetic beads with streptavidin modified on the surface with non-differential sequences; s3, connecting the differential sequence with the non-differential sequence through enzyme linkage reaction; s4, connecting the differential sequence and the differential sequence through enzyme linking reaction. The synthesis method is simple and easy to operate, high in accuracy, good in effect and high in flexibility, and can be applied to single cells or other sequencing technologies.
Description
Technical Field
The invention relates to a preparation method of a biological reagent, in particular to a magnetic bead with a molecular label primer sequence and a preparation method thereof.
Background
Single cell sequencing is a technique for amplifying and sequencing a whole genome or a whole transcriptome by taking a single cell as a unit. In the single cell library building and sequencing process, hundreds and thousands of cells are sequenced at each time, in order to distinguish basic information of each cell, a single cell is separated from a single microbead or magnetic bead with a molecular label in a one-to-one correspondence mode, after the cell is lysed, the molecular label on the microbead or magnetic bead is used, in the single cell reverse transcription and amplification process, different molecular labels are added to each single cell or even each m RNA label, so that the key step of single cell sequencing is realized, and each magnetic bead needs to carry a primer sequence of different labels. Currently, only Barcode oligo Dt Primer ON Beads (with the product number of 120917) of Chemsenes company can be purchased at home, but the bead is not in stock at home, and has the problems of long shelf life, high cost, unstable different batches and the like due to the reservation from abroad, thereby seriously influencing the product research and development progress of the company and consuming the research and development cost of enterprises. The product is the microsphere of Dropseq, the synthesis method needs to be carried out for 12 times of phosphorylation synthesis, a more expensive synthesis instrument is used, the manufacturing power is lower, and the experimental accuracy is reduced.
Disclosure of Invention
The problems to be solved are as follows: aiming at the problems, the invention provides the magnetic bead and the preparation method thereof, the synthesis method is simple and easy to operate, has high accuracy, good effect and high flexibility, and can be applied to single cells or other sequencing technologies.
The technical scheme is as follows:
the invention provides a magnetic bead with a molecular label primer sequence, which is connected with streptavidin through surface modification, and is connected with a DNA barcode sequence on the surface of the magnetic bead.
In a further embodiment, the DNA barcode sequences are divided into differential sequences and non-differential sequences, the non-differential sequences are linked to the surface of the magnetic beads, and the differential sequences are linked to the non-differential sequences.
The further technical scheme is that the non-differential sequence on the surface of the magnetic bead is 1, and the differential sequence contains more than 2 96 differential sequences.
In a further embodiment, the variant sequence comprises 3 variant sequences of 96, and 96 × 96 combinations are possible.
According to a further technical scheme, the magnetic beads are 30 microns.
A preparation method of magnetic beads with molecular label primer sequences comprises the following steps:
s1, synthetic DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences;
s2, connecting magnetic beads with streptavidin modified on the surface with non-differential sequences;
s3, connecting the differential sequence with the non-differential sequence through enzyme linkage reaction;
s4, connecting the differential sequence and the differential sequence through enzyme linking reaction.
The further technical scheme is that the method comprises the following steps: detecting by a single cell transcriptome sequencing mode, namely a chip experiment.
The further technical scheme is that the method comprises the following steps: n pieces of 96 differential sequences of the differential sequences, wherein N is more than or equal to 2; the times of the enzyme-linked reaction in the S3 are M times; and M is N-1 or N.
A preparation method of magnetic beads with molecular label primer sequences comprises the following steps:
s1, synthetic DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences; the non-differential sequence is a magnetic bead-1 primer; the differential sequences are a magnetic bead-2 primer, a magnetic bead-3 primer and a magnetic bead-4 primer; the differential sequences are 3 DNA barcode sequences with 96 differential sequences, namely the DNA barcode sequences with 96 differential sequences are respectively connected with 3 primers; i.e., the differential sequence 96X96X96 sequence possibilities.
S2, connecting the magnetic beads with the surface modified streptavidin with non-differential sequences:
dissolving the primers by using BW Buffer, wherein the final concentration of the magnetic bead-1 primer is 50 mu M, and the final concentration of other primers is 50 mu M;
transferring 1x10^6 magnetic beads into a centrifugal tube, placing the centrifugal tube on a magnetic frame until the liquid is clarified, and discarding the supernatant;
adding 500 μ l of 1X BW buffer, mixing the magnetic beads uniformly, placing the centrifuge tube on a magnetic frame until the liquid is clear, discarding the supernatant, and repeating the step;
adding 720. mu.l of magnetic bead-1 primer solution of X BW buffer, mixing the magnetic beads uniformly, shaking and incubating for 1 hour at room temperature,
adding 500. mu.l of BW buffer, and washing the magnetic beads twice; adding 500 mu l of TE Buffer, and washing the magnetic beads twice;
adding 1ml of TE for resuspension, transferring to a 15ml centrifuge tube, and adding 2.6ml of TE; s3, connecting the differential sequence with the non-differential sequence;
the first enzyme-linked reaction is carried out,
preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-2 primers and complementary primers into each well, and carrying out enzyme-linked reaction according to the specification of T4DNA Ligase, wherein the magnetic beads are 1x10 ^4 magnetic beads;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1 × TE buffer into each 96 wells, mixing the magnetic beads uniformly, placing ninety-six well plates on a magnetic frame until the liquid is clear, and discarding the supernatant; repeating for 10.3 times;
resuspend with 37.2. mu.l TE buffer, collect the magnetic beads in 96 wells in a 15ml centrifuge tube for the next reaction;
s4, connecting non-differential sequences and non-differential sequences through enzyme linkage reaction:
the second enzyme-linked reaction is carried out,
preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-3 primers and 1x10 ^4 magnetic beads into each well, and carrying out enzyme-linked reaction;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1X TE buffer into each well, mixing the magnetic beads uniformly, placing a 96-well plate on a magnetic frame until the liquid is clear, discarding the supernatant, repeating the steps twice;
resuspend with 37.2. mu.l TE buffer, collect the magnetic beads in 96 wells in a 15ml centrifuge tube for the next reaction;
third enzyme-linked reaction
Preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-4 primers and 1x10 ^4 magnetic beads into each well, and carrying out enzyme-linked reaction;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1X TE buffer into each well, mixing the magnetic beads uniformly, placing a 96-well plate on a magnetic frame until the liquid is clear, discarding the supernatant, repeating the steps twice;
re-suspending the magnetic beads in each well by using TE, collecting the magnetic beads in a centrifugal tube of 1.5ml, and gently blowing, sucking and uniformly mixing;
and subpackaging the magnetic beads for later use.
Further, the streptavidin magnetic beads were purchased from suzhou mykoku biotechnology limited.
Has the advantages that:
the invention synthesizes the primer sequences with different labels on the magnetic beads, adopts the mode of ligase connection and synthesizes the primer sequences with different labels on the magnetic beads according to the Hamming distance principle, and has the advantages of low cost, simple operation, rapid synthesis and stable production process. Compared with the prior synthesis method, the method needs 12 times of phosphorylation synthesis, uses a more expensive synthesis instrument, and has lower manufacturing power, but the technical scheme of the invention only needs 2 times of connection reaction, has high accuracy, is simple to operate, does not depend on the synthesis instrument, and has greater superiority in manufacturing process.
The conventional sequence synthesis and PCR synthesis have certain preference and error rate, but the accuracy of the ligase connection method adopted by the invention is higher than that of the conventional method, so that the one-to-one correspondence relationship between the tag sequence and the marker (such as single cell) can be ensured in sequencing, and the sequencing accuracy is ensured.
The technical scheme of the invention has high sequencing accuracy and low cost, can measure sequences of a plurality of cells at one time, and saves the experiment cost. The label is a fixed label, the information of the label library is known according to a design scheme, and compared with a randomly generated Dropseq label, the label is more convenient for later-stage bioinformatics analysis and error correction. In addition, the magnetic beads are used as the substrate of the molecular label, the magnetic beads are more convenient to apply than the microbeads, and the magnetic bead collection can be realized by using a magnetic frame.
The invention can replace Barcode oligo Dt Primer ON Beads (the product number is 120917) of Chemgees company, and effectively solves the problems of high cost, difficult manufacture, unstable batch and the like.
Drawings
FIG. 1 shows the results of fragment analysis (FragmentAnalyzer) in the control WT11AATI full-automatic capillary electrophoresis apparatus in the examples;
FIG. 2 is the results of Fragment analysis (Fragment Analyzer) in the WT12AATI full-automatic capillary electrophoresis apparatus in the examples;
FIG. 3 is the results of Fragment analysis (Fragment Analyzer) in the WT13AATI full-automatic capillary electrophoresis apparatus in the examples;
FIG. 4 shows the results of Fragment analysis (Fragment Analyzer) in the WT14AATI full-automatic capillary electrophoresis apparatus in example;
FIG. 5 shows the result of library construction of control WT 11;
FIG. 6 shows the library construction results of test group WT 12;
FIG. 7 shows the library construction results of experimental group WT 13;
FIG. 8 shows the library construction results of experimental group WT 14;
FIG. 9 is a graph showing the number distribution of WT 11-specific molecular tags in a control group;
FIG. 10 is a graph showing the number distribution of WT 12-specific molecular tags in the experimental group;
FIG. 11 is a graph showing the number distribution of WT 13-specific molecular tags in the experimental group;
FIG. 12 is a graph showing the number distribution of WT 14-specific molecular tags in the experimental group;
FIG. 13 is a schematic diagram of the structure and method of the present invention.
Detailed Description
Examples
Firstly, experimental conditions: the Barcode oligo Dt Primer ON Beads control group of Chemsenes was named WT11
The invention is 3 samples of experimental group named WT12, WT13, WT14 respectively, the preparation method of the experimental group bead is as follows:
first, synthesis of DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences;
secondly, connecting magnetic beads with streptavidin modified surfaces with non-differential sequences;
1 configuration BW Buffer 10ml
2 the primers were dissolved in BW Buffer at a final concentration of 50. mu.M for the bead-1 primers and 50. mu.M for the other primers.
3 transfer 1X10^6 magnetic beads to a 1.5mL centrifuge tube.
4, placing the centrifugal tube on a magnetic frame until the liquid is clear, and discarding the supernatant.
5 adding 500. mu.l of 1X BW buffer, mixing the magnetic beads uniformly, placing the centrifuge tube on a magnetic frame until the liquid is clear, and discarding the supernatant.
6 repeat step 5 twice.
Adding 720. mu.l of 1X BW buffer into 7, uniformly mixing the magnetic beads, uniformly dividing the magnetic beads into 96-well plates, adding 7.5. mu.l of magnetic beads into each well, adding 2.5ul of magnetic bead-1 (96) into each well, and performing shake incubation for 1 hour at room temperature
8, after the incubation is finished, discarding the supernatant,
9 washing the beads 2 times with TE, collecting the beads in the 96 wells, adding 500. mu.l BW buffer, and washing the beads 1 time.
10 add 1ml TE to resuspend the beads.
And thirdly, connecting the differential sequence with the non-differential sequence through enzyme-linked reaction.
11 first enzyme ligation reaction
1) 96 different magnetic bead-2 primers 2.5ul and complementary primers-1 primers 2.5ul were added to a 96 well plate, and the temperature was reduced to 20 ℃ at 0.1 ℃ for 5 minutes in a PCR instrument.
2) After the reaction, 10ul of magnetic beads were added, and a reagent such as T4DNA Ligase was added to carry out an enzymatic ligation reaction according to the instructions of T4DNA Ligase (Biyuntian 7008).
3) After the reaction is finished, the 96-well plate is placed on a magnetic frame, the reaction is waited for 5min until the supernatant is clear, and the supernatant is discarded.
4) Add 50. mu.l of 1 XTE buffer to each 96 well, mix the beads well, place the 96 well plate on a magnetic rack until the liquid is clear, and discard the supernatant.
5) Repeating the step 3) twice.
6) Resuspend with 20. mu.l TE buffer, collect the beads in 96 wells in a 1ml centrifuge tube, wash 2 times with TE, resuspend with 1ml enzyme-free water for the next reaction.
And fourthly, connecting the differential sequence with the non-differential sequence through enzyme-linked reaction.
12 second enzyme ligation reaction
1) 96 different magnetic bead-3 primers 2.5ul and complementary primers-2.5 ul were added to a 96 well plate, and the temperature in the PCR instrument was reduced to 20 ℃ at 0.1 ℃ for 5 minutes at 95 ℃.
2) After the reaction, 10ul of magnetic beads were added, and the ligation reaction was performed according to the T4DNA Ligase (Biyunshi 7008) protocol.
3) After the reaction is finished, the 96-well plate is placed on a magnetic frame, the reaction is waited for 5min until the supernatant is clear, and the supernatant is discarded.
4) Add 50. mu.l of 1 XTE buffer to each 96 wells, mix the beads, place ninety-six well plates on a magnetic rack until the fluid is clear, and discard the supernatant.
5) Repeating the step 4) twice.
6) Resuspend with 20. mu.l TE buffer, collect the magnetic beads in 96 wells in a 1ml centrifuge tube, wash 2 times with TE, resuspend with 1ml enzyme-free water for the next reaction.
13 third enzyme ligation reaction
1) 96 different magnetic bead-4 primers (2.5 ul) and complementary primers (2.5 ul) were added to a 96-well plate, and the temperature was reduced to 20 ℃ at 0.1 ℃ for 5 minutes in a PCR instrument.
2) After the reaction, 10ul of magnetic beads were added, and the ligation reaction was performed according to the T4DNA Ligase (Biyunshi 7008) protocol.
3) After the reaction is finished, placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
4) add 50. mu.l of 1 XTE buffer to each 96 wells, mix the beads, place ninety-six well plates on a magnetic rack until the fluid is clear, and discard the supernatant.
5) Repeating the step 4) twice.
6) Resuspend with 20. mu.l TE buffer, collect the beads in 96 wells in a 1ml centrifuge tube, wash 2 times with TE, wash 2 times with PBST (0.1%), and store in 1ml PBST.
After each magnetic bead is connected with a first non-differential sequence, 3 96 differential sequences are connected in an enzyme-linked mode for 3 times, aiming at the current experiment, 96X96 differential sequences can be used as 2 differential sequences and can mark 96X96 single cells, but 3 differential sequences can generate more differences, 96X96X96 magnetic beads prepared by the method can mark more single cells theoretically.
Second, verification experiment
1 materials of the experiment
Cell: experimental conditions of K562 cell line: the control group of Barcode oligo Dt Primer ONBeads from Chemmenes was named WT 11.
The invention is that 3 samples of the experimental group are respectively named as WT12, WT13 and WT14
2. Laboratory apparatus
3. Experimental reagent
| Name (R) | Manufacturer of the product | |
| Nuclease-Free Water | Beyotime | |
| Anhydrous ethanol | Tianjin Damao | |
| Tris,PH7.5 | Lovery forest | |
| EDTA PH8 | Lovery forest | |
| Nacl | Lovery forest | |
| DTT | Tiangen (root of heaven) | |
| RT Buffer | Tiangen (root of heaven) | |
| RT | Tiangen (root of heaven) | |
| Ficoll PM400 | Worker of ordinary skill | |
| RNAse Inhibitor | Novozan/Aibotaike | |
| TS1 | Worker of ordinary skill | |
| PCR Buffer | KAPA | |
| dNTPs | Worker of ordinary skill | |
| SP1 | Worker of ordinary skill | |
| Tips LTS 200UL Filter RT-L200FLR | Rainin | |
| DNA LoBind Tubes,1.5ml | Eppendorf | |
| TempAssure PCR8-tube | USA Scientific | |
| 10 mu L low adsorption box-packed | Axygen | |
| 200 mu L low adsorption box-packed | Axygen | |
| 1000 mu L boxed gun head | Axygen |
4 chip experiment
4.1 treating cells
(1) K562 cell treatment
a. The cell morphology was observed under a microscope, and the cells floated in the medium in a round shape. The cell suspension was aspirated into a 15mL sterile centrifuge tube with a Pasteur pipette at 1300rpm for 3min, the supernatant was discarded, and 800-.
b. And (3) measuring the concentration: 10uL trypan blue +10uL cells, pipetted and mixed 10 times. 10uL were counted on a cytometer.
c. Diluting the cell concentration, namely diluting the cell suspension into 1 × 10 cells/mL to be used.
4.2 chip experiments
1 preparing an experimental chip, adding 80ul (1X10^5) of cells, adding 8 ten thousand beads (microspheres) after the cells are fully paved on the chip, and cracking the cells. The mRNA was captured at room temperature for about 1 hour. Then, the magnetic beads are collected and subjected to reverse transcription and transcriptional gene amplification.
4.3 measure the value of Qubit
4.4AATI full-automatic capillary electrophoresis Fragment analysis (Fragment Analyzer) detection
(1) Running the software "Fragment An1 yzer"
(2) Analytical software "Prosize 2.0"
FIGS. 1-4; in the figure, the ordinate RFU is an abbreviation for relative fluorescence units, and the abscissa indicates the fragment size in bp.
At present, we need fragment analysis peak size around 1000-2000 bp. As can be seen, the magnetic beads of the present invention have similar fragment results compared to the Dropseq beads, and meet the standard for single cell transcriptome banking.
4.5 library construction TruePrepTM Index Kit V2for according to the Kit
(VazymeTD502) library construction. The results are shown in FIGS. 5-8, which show that the size of the Library fragments is consistent when the primers of Barcode oligo Dt primer Beads from Chemmenes are used to perform reverse transcription and amplification on mRNA (TruePrep DNA Library V2for illumina TD502), and the experimental conditions of the present invention are the same. And (4) analyzing results: and (4) performing on-machine sequencing when the library building result meets the experimental standard.
4.6 bioinformatics results analysis
1) Quality control analysis of molecular tags
The original data can extract the ratio of a difference sequence, namely a molecular label (barcode), namely the ratio of reads with the number of cellbarcode reads larger than 1;
the said barcode: molecular tags, one for each magnetic bead (differential sequence), are used to label single cells. Each sequence sequenced was read and the multiple sequences were reads. The UMI is a random sequence behind a molecular tag sequence and can mark different mRNA and genes.
2 sample comparison analysis
3 cell number assessment and UMI statistics
In the above table, a represents a sample name (Samplename); b represents the cell number (Cells number); c represents the proportion of the detection sequence evaluated as Cells (Cells reads PCT); d represents the sequencing Saturation, the ratio of gene UMI repeats (Saturation); e represents the average number of sequencing assays per Cell (Mean Reads/Cell); f represents the Median number of UMIs for cells (Median UMI); g represents the Total number of Genes identified (Total Genes); h represents the Median number of Genes in the cell (medians Genes).
The UMI is a random sequence behind a molecular tag sequence and can mark different mRNA and genes.
4UMI number distribution is shown in FIGS. 9-12
Conclusion analysis: from the analysis of bioinformatics results, the magnetic Beads prepared by the invention can achieve the same analysis effect in single cell analysis as Barcode oligo Dt Primer ON Beads products of Chemgeenes company, and even from the filtered data and the comparison data, the invention has certain superiority in the single cell transcriptome sequencing.
Claims (9)
1. The magnetic bead with the molecular tag primer sequence is characterized in that the surface of the magnetic bead is modified and connected with streptavidin, and the surface of the magnetic bead is connected with a DNA barcode sequence.
2. The magnetic bead with a molecular tag primer sequence of claim 1, wherein the DNA barcode sequences are differentiated sequences and non-differentiated sequences, the non-differentiated sequences are linked to the surface of the magnetic bead, and the differentiated sequences are linked to the non-differentiated sequences.
3. The magnetic bead of claim 2, wherein the non-discriminatory sequence on the surface of the magnetic bead is 1 and the discriminatory sequence comprises more than 2 and 96 discriminatory sequences.
4. The magnetic bead with molecular tag primer sequences of claim 3, wherein said divergent sequences comprise 3 96 divergent sequences.
5. The magnetic bead with the molecular tag primer sequence of claim 1, wherein the magnetic bead is 30 μm in size.
6. A preparation method of magnetic beads with molecular label primer sequences is characterized by comprising the following steps:
s1, synthetic DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences;
s2, connecting magnetic beads with streptavidin modified on the surface with non-differential sequences;
s3, connecting the differential sequence with the non-differential sequence through enzyme linkage reaction;
s4, connecting the differential sequence and the differential sequence through enzyme linking reaction.
7. The method of claim 6, wherein the step of detecting comprises the steps of: detection is carried out by single cell transcriptome sequencing mode.
8. The method of claim 7, comprising the steps of: the differential sequences are N96 differential sequences, and N is more than or equal to 2; the times of the enzyme-linked reaction in the S3 are M times; and M is N-1 or N.
9. The method of claim 6, comprising the steps of:
s1, synthetic DNA barcode sequence: respectively synthesizing differential sequences and non-differential sequences; the non-differential sequence is a magnetic bead-1 primer; the differential sequences are a magnetic bead-2 primer, a magnetic bead-3 primer and a magnetic bead-4 primer;
s2, connecting the magnetic beads with the surface modified streptavidin with non-differential sequences:
dissolving the primers by using BW Buffer, wherein the final concentration of the magnetic bead-1 primer is 50 mu M, and the final concentration of other primers is 50 mu M;
transferring 1x10^6 magnetic beads into a centrifugal tube, placing the centrifugal tube on a magnetic frame until the liquid is clarified, and discarding the supernatant;
adding 500 μ l of 1X BW buffer, mixing the magnetic beads uniformly, placing the centrifuge tube on a magnetic frame until the liquid is clear, discarding the supernatant, and repeating the step;
adding 720. mu.l of magnetic bead-1 primer solution of X BW buffer, mixing the magnetic beads uniformly, shaking and incubating for 1 hour at room temperature,
adding 500. mu.l of BW buffer, and washing the magnetic beads twice; adding 500 mu l of TE Buffer, and washing the magnetic beads twice;
adding 1ml of TE for resuspension, transferring to a 15ml centrifuge tube, and adding 2.6ml of TE; s3, connecting the differential sequence with the non-differential sequence;
the first enzyme-linked reaction is carried out,
preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-2 primers and complementary primers into each well, and carrying out enzyme-linked reaction according to the specification of T4DNA Ligase, wherein the magnetic beads are 1x10 ^4 magnetic beads;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1 × TE buffer into each 96 wells, mixing the magnetic beads uniformly, placing ninety-six well plates on a magnetic frame until the liquid is clear, and discarding the supernatant; repeating for 10.3 times;
resuspend with 37.2. mu.l TE buffer, collect the magnetic beads in 96 wells in a 15ml centrifuge tube for the next reaction;
s4, connecting non-differential sequences and non-differential sequences through enzyme linkage reaction:
the second enzyme-linked reaction is carried out,
preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-3 primers and 1x10 ^4 magnetic beads into each well, and carrying out enzyme-linked reaction;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1X TE buffer into each well, mixing the magnetic beads uniformly, placing a 96-well plate on a magnetic frame until the liquid is clear, discarding the supernatant, repeating the steps twice;
resuspend with 37.2. mu.l TE buffer, collect the magnetic beads in 96 wells in a 15ml centrifuge tube for the next reaction;
third enzyme-linked reaction
Preparing an enzyme-linked reagent in a 96-well plate, adding 96 different magnetic bead-4 primers and 1x10 ^4 magnetic beads into each well, and carrying out enzyme-linked reaction;
placing the 96-well plate on a magnetic frame, waiting for 5min until the supernatant is clear, and discarding the supernatant;
adding 50 μ l of 1X TE buffer into each well, mixing the magnetic beads uniformly, placing a 96-well plate on a magnetic frame until the liquid is clear, discarding the supernatant, repeating the steps twice;
re-suspending the magnetic beads in each well by using TE, collecting the magnetic beads in a centrifugal tube of 1.5ml, and gently blowing, sucking and uniformly mixing;
and subpackaging the magnetic beads for later use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811653760.7A CN111378728A (en) | 2018-12-31 | 2018-12-31 | Magnetic bead with molecular label primer sequence and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811653760.7A CN111378728A (en) | 2018-12-31 | 2018-12-31 | Magnetic bead with molecular label primer sequence and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111378728A true CN111378728A (en) | 2020-07-07 |
Family
ID=71219666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811653760.7A Pending CN111378728A (en) | 2018-12-31 | 2018-12-31 | Magnetic bead with molecular label primer sequence and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111378728A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251504A (en) * | 2020-09-09 | 2021-01-22 | 新格元(南京)生物科技有限公司 | Magnetic microsphere with molecular label sequence and preparation method thereof |
| CN113106086A (en) * | 2021-04-15 | 2021-07-13 | 上海烈冰生物医药科技有限公司 | Preparation method of magnetic beads with DNA labels |
| WO2022133734A1 (en) * | 2020-12-22 | 2022-06-30 | Singleron (Nanjing) Biotechnologies, Ltd. | Methods and reagents for high-throughput transcriptome sequencing for drug screening |
| WO2022188827A1 (en) * | 2021-03-10 | 2022-09-15 | Nanjing University | Chemical sample indexing for high-throughput single-cell analysis |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498040A (en) * | 2016-10-12 | 2017-03-15 | 浙江大学 | A kind of molecular labeling microballon and the unicellular sequence measurement of the high flux based on the molecular labeling microballon |
| WO2017124101A2 (en) * | 2016-01-15 | 2017-07-20 | The Broad Institute Inc. | Semi-permeable arrays for analyzing biological systems and methods of using same |
| WO2017168329A1 (en) * | 2016-03-28 | 2017-10-05 | Boreal Genomics, Inc. | Droplet-based linked-fragment sequencing |
| CN107873054A (en) * | 2014-09-09 | 2018-04-03 | 博德研究所 | The method and apparatus based on droplet for compound single-cell nucleic acid analysis |
| CN108350497A (en) * | 2015-08-28 | 2018-07-31 | Illumina公司 | Unicellular nucleic acid sequence analysis |
-
2018
- 2018-12-31 CN CN201811653760.7A patent/CN111378728A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107873054A (en) * | 2014-09-09 | 2018-04-03 | 博德研究所 | The method and apparatus based on droplet for compound single-cell nucleic acid analysis |
| CN108350497A (en) * | 2015-08-28 | 2018-07-31 | Illumina公司 | Unicellular nucleic acid sequence analysis |
| WO2017124101A2 (en) * | 2016-01-15 | 2017-07-20 | The Broad Institute Inc. | Semi-permeable arrays for analyzing biological systems and methods of using same |
| WO2017168329A1 (en) * | 2016-03-28 | 2017-10-05 | Boreal Genomics, Inc. | Droplet-based linked-fragment sequencing |
| CN106498040A (en) * | 2016-10-12 | 2017-03-15 | 浙江大学 | A kind of molecular labeling microballon and the unicellular sequence measurement of the high flux based on the molecular labeling microballon |
Non-Patent Citations (2)
| Title |
|---|
| FAN ZHANG等: "Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube", 《NATURE BIOTECHNOLOGY》 * |
| XIAOPING HAN等: "Mapping the Mouse Cell Atlas by Microwell-Seq", 《CELL》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251504A (en) * | 2020-09-09 | 2021-01-22 | 新格元(南京)生物科技有限公司 | Magnetic microsphere with molecular label sequence and preparation method thereof |
| WO2022133734A1 (en) * | 2020-12-22 | 2022-06-30 | Singleron (Nanjing) Biotechnologies, Ltd. | Methods and reagents for high-throughput transcriptome sequencing for drug screening |
| WO2022188827A1 (en) * | 2021-03-10 | 2022-09-15 | Nanjing University | Chemical sample indexing for high-throughput single-cell analysis |
| CN113106086A (en) * | 2021-04-15 | 2021-07-13 | 上海烈冰生物医药科技有限公司 | Preparation method of magnetic beads with DNA labels |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111378728A (en) | Magnetic bead with molecular label primer sequence and preparation method thereof | |
| US20220162673A1 (en) | Methods for preparative in vitro cloning | |
| US8802367B2 (en) | Methods for quantitative cDNA analysis in single-cell | |
| US9752176B2 (en) | Methods for preparative in vitro cloning | |
| JP6404714B2 (en) | Multivariate diagnostic assay and method for using the same | |
| CA2606723A1 (en) | Multiplex capture of nucleic acids | |
| EP4095256A1 (en) | Droplet microfluidics-based single cell sequencing and applications | |
| CN105986324A (en) | Construction method and application of cyclic small RNA library | |
| CN111549025B (en) | Strand displacement primer and cell transcriptome library construction method | |
| US20240043919A1 (en) | Method for traceable medium-throughput single-cell copy number sequencing | |
| CN107447031B (en) | Mutant nucleic acid digital analysis method for loop-mediated isothermal amplification in emulsion | |
| JP5906306B2 (en) | CDNA amplification method derived from a small amount of sample | |
| US11718872B2 (en) | Method for obtaining single-cell mRNA sequence | |
| WO2022188827A1 (en) | Chemical sample indexing for high-throughput single-cell analysis | |
| US20110237449A1 (en) | Methods and Compositions for Nucleic Acid Purification | |
| JP5519304B2 (en) | Method for uniformly amplifying nucleic acid and highly sensitive detection method | |
| CN114096679B (en) | Nucleic acid amplification method using solid phase carrier | |
| CN110878342A (en) | Digital absolute quantitative analysis method for nucleic acid marker based on single fluorescent particle counting of flow cytometer | |
| EP4347870A1 (en) | Methods and systems for determining cell-cell interaction | |
| CN117089597A (en) | Single cell library construction sequencing method and application thereof | |
| CN117166064A (en) | Method, primer set and kit for constructing target library |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200707 |