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CN113215233A - Microsphere with specific oligonucleotide sequence for single cell sequencing - Google Patents

Microsphere with specific oligonucleotide sequence for single cell sequencing Download PDF

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CN113215233A
CN113215233A CN202110531839.8A CN202110531839A CN113215233A CN 113215233 A CN113215233 A CN 113215233A CN 202110531839 A CN202110531839 A CN 202110531839A CN 113215233 A CN113215233 A CN 113215233A
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郑文山
裴颢
汤王鋆
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Mezhuo Biotechnology Zhejiang Co ltd
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Abstract

The invention belongs to a single cell sequencing related reagent, and particularly relates to a polyacrylamide microsphere with a specific oligonucleotide sequence and a preparation method thereof. The invention takes polyacrylamide microspheres as the substrate of oligonucleotide sequence molecular tags, and three sections of oligonucleotides (oligos) are connected to the polyacrylamide microspheres in sequence by using a chemical bond and enzyme connection method to synthesize the polyacrylamide microspheres with unique oligo sequences. The polyacrylamide microsphere has a specific nucleotide sequence and a larger volume, is customized according to the size of a common microfluidic chip flow channel on the market, can independently fill one flow channel and be arranged orderly in practical use due to the slightly soft and micro-elastic texture of the polyacrylamide microsphere, obviously reduces the difficulty and batch difference of the single microsphere wrapped by liquid drops, is more suitable for single cell sequencing based on droplet microfluidic than the traditional hard microsphere, improves the stability and accuracy of a single cell sequencing result, and can be widely applied to the research of medicine, biology and the like.

Description

Microsphere with specific oligonucleotide sequence for single cell sequencing
Technical Field
The invention belongs to a single cell sequencing reagent, and particularly relates to a polyacrylamide microsphere with a specific oligonucleotide sequence.
Background
Single-cell RNA-sequencing (scRNA-seq) is a new technology which is started in recent years, can obtain a full transcriptome expression profile from a single cell level, and can perform high-throughput sequencing after amplification, so that the gene expression level in the single cell can be efficiently detected, and the single-cell RNA-sequencing has important application values in the fields of diagnosis and treatment of tumors, design of targeted drugs, development and differentiation of stem cells and the like.
Single cell transcriptome sequencing can sequence hundreds of thousands of cells simultaneously because each cell is tagged with a different tag, and each mRNA is similarly assigned a unique molecular tag in order to distinguish each mRNA in each cell. The current micro-fluidic platform for single cell sequencing is mainly characterized in that a single cell and a single microsphere with a molecular label and a cell label are placed in a micropore environment, after the cell is cracked, different cells can carry specific cell labels, and different mRNA of the same cell is marked by different molecular labels through reverse transcription. The method can track the cell source of each gene, can quantify mRNA, obviously improves sequencing flux, reduces sequencing cost and lightens the influence of amplification preference. However, the latest single cell sequencing based on the droplet microfluidic technology exists at present, complex multiple steps can be integrated on one microfluidic chip, the traditional microspheres with molecular labels have the problems of high manufacturing cost, difficulty in preparation, low preparation success rate, unstable effects of different batches and the like, the most serious reason is that the droplet microfluidic technology is difficult to form single packages on the traditional microspheres, the stability and the accuracy of single cell sequencing results are influenced, and meanwhile, the single cell sequencing cost is high, and the method can not be widely applied.
Disclosure of Invention
The invention aims to solve the problems, and aims to provide a large-volume non-hard polyacrylamide microsphere with a specific oligonucleotide sequence, wherein the polyacrylamide microsphere is provided with a fixed molecular tag sequence, and the preparation process is simple, the batch difference is small, the error rate is low, and the cost is low.
In order to achieve the purpose, the invention adopts the following technical scheme:
the embodiment of the invention discloses a large-volume non-rigid polyacrylamide microsphere with an oligonucleotide sequence.
The surface of the polyacrylamide microsphere is sequentially modified and linked with three or more oligonucleotide sequences, wherein the oligonucleotide sequences comprise three segments, D1, D2 and D3.
The sequence of D1 is: ACACTCTTTCCCTACACGACGCTCTTCCGATCT [ X ] A; the sequence of D2 is CTG [ Y ] A, and the sequence of D3 is CTG [ Z ] polyT.
Wherein X, Y, Z represents DNA sequences of 4-14bp length, and polyT represents 10-35T.
Connecting the three segments by chemical bonds and DNA ligase or polymerase, connecting the three segments to polyacrylamide microspheres, and synthesizing the polyacrylamide microspheres with oligo sequences.
The polyacrylamide microsphere also contains auxiliary sequences RC2 and RC 3; RC2 is the complementary sequence of ACTG [ Y ] and RC3 bits [ Z ] and [ polyT ].
Further, X represents an arbitrary DNA sequence 6-12bp long;
further, Y represents an arbitrary DNA sequence 6 to 12bp long;
further, Z represents an arbitrary DNA sequence 6-12bp long.
Further, polyT represents 15-25 Ts.
The invention also provides a method for synthesizing microspheres with specific oligonucleotide sequences, which comprises the following steps:
(1) synthesizing three oligonucleotide sequences D1, D2 and D3 and corresponding auxiliary sequences RC2 and RC3 respectively;
(2) preparing polyacrylamide microspheres by using an oligonucleotide sequence D1, polyacrylamide microsphere monomers and an initiator through a microfluidic technology to obtain a polyacrylamide microsphere mixture A connected with D1;
(3) adding DNA ligase into the polyacrylamide microsphere mixture A obtained in the step (2) to obtain a mixed system M; mixing the oligonucleotide sequence D2 and the auxiliary sequence RC2 with a mixing system M to obtain a polyacrylamide microsphere mixture B connected with D1 and D2;
(4) and (3) adding DNA ligase into the polyacrylamide microsphere mixture B obtained in the step (3) to obtain a mixed system N, mixing the oligonucleotide sequence D3 and the auxiliary sequence RC3 with the mixed system N to obtain polyacrylamide microspheres connected with D1, D2 and D3, and collecting and re-suspending the polyacrylamide microspheres in a water phase to obtain the polyacrylamide microspheres with molecular tag sequences. Furthermore, the diameter of the microsphere is 30-300 microns as compared with the size of a flow channel commonly used by a microfluidic chip in the market, so that the flow channel is easily occupied, and liquid drops can be arranged in the flow channel in order and in a single column. The polyacrylamide microsphere has a specific nucleotide sequence and has the following beneficial effects:
1. the volume is large, the micro-fluidic chip is customized according to the size of a common micro-fluidic chip flow channel on the market, and the stability is high; 2. because the polyacrylamide microspheres are soft and slightly elastic, one flow channel can be occupied independently and arranged orderly in practical use, and the blockage in the flow channel is less prone to be caused than that caused by hard materials; 3. the difficulty of wrapping a single microsphere by a droplet is obviously reduced, so that the microsphere is more suitable for single cell sequencing based on droplet microfluidics than the traditional hard small microsphere, the single wrapping rate is more than 80%, and the stability and the accuracy of a single cell sequencing result are improved; 4. the surface of the polyacrylamide microsphere is modified and linked with three or more different oligonucleotide sequences, so that the microsphere has a plurality of varieties.
Drawings
FIG. 1 is a schematic diagram of polyacrylamide microsphere sequencing; comprises the following steps in sequence: the method comprises the following steps of single cell sequencing target cells 1, polyacrylamide microspheres 2 with oligonucleotide sequences, a mixed solution 3 containing cell lysate, oil 4 with a surfactant, liquid drops 5 successfully and singly wrapping the target cells and the polyacrylamide microspheres with the oligonucleotide sequences, and a liquid drop collection outlet 6.
FIG. 2 is a photomicrograph of polyacrylamide microspheres.
FIG. 3 is a fluorescent microscope photograph of polyacrylamide microspheres.
Fig. 4 is a size distribution diagram of polyacrylamide microspheres.
Fig. 5 is a picture of a droplet after polyacrylamide microsphere is coated.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.
EXAMPLES preparation of Polyacrylamide microspheres
Materials: reagent: t4 ligase and 10 × Ligation Buffer were purchased from NEB; taq DNA polymerase was purchased from Takara, 40% acrylamide solution, ammonium persulfate, N, N, N ', N' -tetramethylethylenediamine was purchased from Sigma; 1M Tris-HCI (pH 8.0), 5M NaCl, acrylamide/bisacrylamide 19:1 (40% (w/v) solution), DNase/RNase-Free Water were purchased from Saimer fly; droplet Generation Oil for probes purchased from BioRad; TE (TE buffer) was purchased from Biotechnology engineering (Shanghai) Ltd; HFE 7100 was purchased from 3M.
1. Preparation of oligonucleotides:
the sequence of D1 is: ACACTCTTTCCCTACACGACGCTCTTCCGATCT [ X ] A;
the sequence of D2 is: CTG [ Y ] A;
the sequence of D3 is: CTG [ Z ] TTTTTTTTTTTTTTTTTTTT;
the order of RC2 is: ACTG [ Y ] complement to ACTG;
the order of RC3 is: (Z) the complement of TTTTTTTTTTTTTTTTTTT;
the above X, Y, Z sequence represents an arbitrary DNA sequence of 4-14bp in length. For experimental convenience, in this example, we selected 96 sequences for illustration, and the specific sequences of the 96 sequences are:
Figure BDA0003067629160000041
Figure BDA0003067629160000051
Figure BDA0003067629160000061
the oligonucleotide sequence used was dissolved in TE buffer at a final concentration of 100 uM. D2 and RC2, D3 and RC3 should anneal the single strand to double strands prior to subsequent ligation reactions.
Mixing 100 μ M D2 and 100 μ M RC2 in equal volume, heating the mixed system to 94 deg.C for 2min, slowly cooling to obtain 50uM D2-RC2 solution, and storing at-20 deg.C (long-term) or 4 deg.C (short-term).
Mixing 100 μ M D3 and 100 μ M RC3 in equal volume, heating the mixed system to 94 deg.C for 2min, slowly cooling to obtain 50uM D3-RC3 solution, and storing at-20 deg.C (long-term) or 4 deg.C (short-term).
2. Preparation of polyacrylamide microspheres
(1) First step ligation reaction
25uL of 100uM D1 were dispensed into 96-well plates, one D1 sequence per well. To each well was added 17uL of water, 0.2uL of 1M Tris-HCI (pH 8.0), 0.2uL of 5M NaCl, 4.5uL of 40% aqueous acrylamide solution, 3.1uL of acrylamide/bisacrylamide 19:1 (40% (w/v) solution), 1mg of ammonium persulfate. An Oil phase solution was prepared with 20uL of N, N, N ', N' -tetramethylethylenediamine dissolved in 20mL of Droplet Generation Oil for probes. For each aqueous solution in the 96-well plate, 200uL of oil phase solution and a microfluidic chip are used to generate droplets, and the diameter of the droplets is controlled to be 30-300 um. The first ligation reaction was completed by mixing the droplets from the 96 well plates and allowing them to stand at 65 ℃ for 8 hours or more. The reaction product was washed five times with HFE 7100, and the upper aqueous phase was retained for each wash. Then, five TE buffer washes were performed, and the lower gel-like product remained upon washing. So as to obtain the polyacrylamide microsphere connected with the D1 sequence, which can be used for the next reaction.
(2) Second step ligation reaction
In each well of a 96-well plate, 10uL of the product in (1) was added. An additional 20uL of 50uM D2-RC2 solution was added, one for each well, D2-RC2 sequence. Then 2.5uL of T4DNA ligase and 5. mu. L T4DNA ligase Buffer (10X) are added into each well, 12.5uL of water is added, the mixture is mixed evenly and placed in a PCR instrument for reaction, the program is 25 ℃ for 20min, ice is used for 1h, and the mixture is kept at 4 ℃, thus completing the second ligation reaction. The reaction products in the 96-well plate are combined, and after five times of TE buffer solution washing, polyacrylamide microspheres connected with D2 and D1 sequences are obtained and can be used for the next reaction.
(3) Third step of ligation
In each well of a 96-well plate, 10uL of the product in (2) was added. An additional 20uL of 50uM D3-RC3 solution was added, one for each well, D3-RC3 sequence. Then 2.5uL of T4DNA ligase and 5 mu L T4DNA ligase Buffer (10X) are added into each hole, 12.5uL of water is added, the mixture is uniformly mixed and then placed in a PCR instrument for reaction, the procedure is that a hot cover is arranged at 105 ℃, the temperature is 25 ℃ for 20min, the mixture is kept on ice for 1h, and the third step of ligation reaction is completed after 4 ℃. The reaction product was washed five times with TE buffer to obtain polyacrylamide microspheres linked to the sequences D3, D2 and D1.
In this embodiment, polyacrylamide microspheres having oligonucleotide sequences (hereinafter referred to as "microspheres") are aligned in a flow channel in a single row, so that the rate of entry of a micro-flow from a branch flow channel into a main flow channel can be controlled, and the probability of single encapsulation of the microspheres can be increased. The single-encapsulation rate (i.e. the ratio of droplets containing only one microsphere to the total number of droplets) is calculated by taking a picture of the obtained droplets under a microscope, and then calculating the number of droplets containing only one microsphere and the total number of all droplets, wherein the ratio of the former to the latter is the single-encapsulation rate, i.e. the encapsulation rate is the number of successful single-encapsulated particle droplets/the total number of droplets. Calculated according to fig. 5, the encapsulation efficiency of the microspheres prepared in this example was 88%.
The encapsulation rate of the microspheres is far lower than the encapsulation rate of 20 percent of the traditional droplet microfluidic technology (such as dropseq) by using a magnetic bead microsphere method.
The particle size distribution of the microspheres prepared by the method is 30-300 mu m, and the particle size of the microspheres can be adjusted according to actual needs. The microspheres prepared in this example have uniform diameter and size distribution of 40-54 μm, and a coefficient of variation CV of size of 5% (as shown in FIG. 4).
In the embodiment of the invention, the polyacrylamide material is tough, soft and slightly elastic, and is less prone to blocking a micro-channel than hard microspheres.
In the embodiment of the invention, the target detection cell sap is diluted, so that the speed of the target cells entering the main flow channel from the branch flow channel is slower than the speed of the microspheres entering the main flow channel, thereby realizing the single cell encapsulation of liquid drops.
In the embodiment of the invention, the flow rate of the oil phase is controlled to control the size of the liquid drop, so that the probability that the target cell and the microsphere are simultaneously and singly wrapped by the liquid drop is controllable. If the droplet is too small, the probability that the target cell and the microsphere are simultaneously covered is low, and if the droplet is too large, the probability that the target cell and the microsphere are simultaneously covered is also low, and it may be difficult to generate a droplet.
The polyacrylamide microsphere with the oligonucleotide sequence eliminates the increase of single wrapping difficulty caused by the difficulty in controlling the speed of the small-volume microsphere entering the main flow channel in a microflow, and compared with a hard microsphere, the polyacrylamide is tough, soft and slightly elastic and is less prone to blocking the microflow channel.
In conclusion, the polyacrylamide microspheres have larger volume, are customized according to the size of a common microfluidic chip flow channel on the market, and can independently fill one flow channel and be arranged orderly in practical use due to the slightly soft and micro-elastic texture of the polyacrylamide microspheres, so that the difficulty of wrapping a single microsphere by a liquid drop is obviously reduced, the microspheres are more suitable for single cell sequencing based on the droplet microfluidic than the traditional hard microspheres, and the stability and the accuracy of a single cell sequencing result are improved.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; it is intended that the following claims be interpreted as including all such alterations, modifications, and equivalents as fall within the true spirit and scope of the invention.

Claims (10)

1. The polyacrylamide microsphere with a specific oligonucleotide sequence is characterized in that the surface of the polyacrylamide microsphere is sequentially modified and linked with three oligonucleotide sequences, D1, D2 and D3;
wherein the sequence of D1 is: ACACTCTTTCCCTACACGACGCTCTTCCGATCT [ X ] A;
the sequence of D2 is CTG (Y) A;
the sequence of D3 is CTG [ Z ] [ polyT ];
wherein X, Y, Z represents DNA sequences of 4-14bp length, and polyT represents 10-35T;
the three oligonucleotide sequences are linked to DNA ligase or polymerase by chemical bonds.
2. The polyacrylamide microsphere of claim 1, wherein said polyacrylamide microsphere further comprises the auxiliary sequences RC2, RC 3;
RC2 is a complementary sequence of ACTG [ Y ] ACTG, RC3 is a complementary sequence of [ Z ] polyT ];
RC2 and RC3 are attached to the microsphere structure using chemical bonds and DNA ligase or polymerase.
3. The polyacrylamide microsphere of claim 1, wherein the structure of the polyacrylamide microsphere is a microporous solid sphere structure, and the microsphere is soft, tough and slightly elastic and is less prone to blockage in a flow channel than a hard material.
4. The polyacrylamide microsphere of claim 1, wherein the polyacrylamide microsphere can independently fill one flow channel and is arranged orderly, the time interval between the microsphere and the subsequent flow channel is constant, and the microsphere can be singly wrapped by controlling the speed of the microsphere entering the main flow channel.
5. The polyacrylamide microsphere of claim 1, wherein X represents any DNA sequence 6-12bp long.
6. The polyacrylamide microsphere of claim 1 wherein Y represents any DNA sequence 6-12bp long.
7. The polyacrylamide microsphere of claim 1 wherein Z represents any DNA sequence 6-12bp long.
8. The polyacrylamide microsphere of claim 1 wherein polyT represents 15-25 Ts.
9. The polyacrylamide microsphere of claim 1, wherein the diameter of said polyacrylamide microsphere is between 30-300 μm.
10. A method for preparing polyacrylamide microspheres with specific oligonucleotide sequences as defined in claim 1, comprising the steps of:
(1) synthesizing three oligonucleotide sequences D1, D2 and D3 and corresponding auxiliary sequences RC2 and RC3 respectively;
(2) preparing polyacrylamide microspheres by using an oligonucleotide sequence D1, polyacrylamide microsphere monomers and an initiator through a microfluidic technology to obtain a polyacrylamide microsphere mixture A connected with D1;
(3) adding DNA ligase into the polyacrylamide microsphere mixture A obtained in the step (2) to obtain a mixed system M; mixing the oligonucleotide sequence D2 and the auxiliary sequence RC2 with a mixing system M to obtain a polyacrylamide microsphere mixture B connected with D1 and D2;
(4) adding DNA ligase into the polyacrylamide microsphere mixture B obtained in the step (3) to obtain a mixed system N, mixing the oligonucleotide sequence D3 and the auxiliary sequence RC3 with the mixed system N to obtain polyacrylamide microspheres connected with D1, D2 and D3, and collecting and re-suspending the polyacrylamide microspheres in a water phase to obtain polyacrylamide microspheres with molecular tag sequences;
preferably, steps (2), (3) and (4) are carried out in a microplate.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242060A1 (en) * 2021-05-15 2022-11-24 墨卓生物科技(浙江)有限公司 Microsphere with specific oligonucleotide sequence for single-cell sequencing
CN117496274A (en) * 2023-12-29 2024-02-02 墨卓生物科技(浙江)有限公司 Classification counting method, system and storage medium based on liquid drop images

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925572A (en) * 2016-06-07 2016-09-07 厦门大学 DNA encoding microsphere and synthetic method thereof
CN107075543A (en) * 2014-04-21 2017-08-18 哈佛学院院长及董事 System and method for bar coded nucleic acid
CN108169129A (en) * 2017-12-20 2018-06-15 中国科学院沈阳应用生态研究所 A kind of method for realizing microballoon single-row sample introduction
CN109851711A (en) * 2018-12-29 2019-06-07 苏州绘真生物科技有限公司 Soluble hydrogel microsphere and preparation method thereof and the application in Single cell analysis
CN111073791A (en) * 2018-10-19 2020-04-28 深圳华大生命科学研究院 Particle focusing chip, single cell preparation system and single cell preparation method
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
CN113814008A (en) * 2020-08-28 2021-12-21 上海交通大学 Microfluidic channel structure, chip, particle ordered arrangement method and application

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100564618C (en) * 2007-06-13 2009-12-02 北京万达因生物医学技术有限责任公司 Molecule substitution label sequencing parallel detection method-oligomictic nucleic acid code label molecule library micro-sphere array analysis
EP3619307B1 (en) * 2017-05-05 2021-03-24 The United States of America, as represented by the Secretary, Department of Health and Human Services Methods of preparing a re-usable single cell and methods for analyzing the epigenome, transcriptome, and genome of a single cell
CN110241178B (en) * 2019-06-25 2023-03-31 北京博奥晶方生物科技有限公司 Single-cell transcriptome sequencing high-throughput rapid library preparation method and detection kit
CN113234718B (en) * 2021-05-15 2023-01-06 上海墨卓生物科技有限公司 Double-layer microsphere with oligonucleotide sequence for single cell sequencing
CN113215233A (en) * 2021-05-15 2021-08-06 墨卓生物科技(浙江)有限公司 Microsphere with specific oligonucleotide sequence for single cell sequencing

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107075543A (en) * 2014-04-21 2017-08-18 哈佛学院院长及董事 System and method for bar coded nucleic acid
CN105925572A (en) * 2016-06-07 2016-09-07 厦门大学 DNA encoding microsphere and synthetic method thereof
CN108169129A (en) * 2017-12-20 2018-06-15 中国科学院沈阳应用生态研究所 A kind of method for realizing microballoon single-row sample introduction
CN111073791A (en) * 2018-10-19 2020-04-28 深圳华大生命科学研究院 Particle focusing chip, single cell preparation system and single cell preparation method
CN109851711A (en) * 2018-12-29 2019-06-07 苏州绘真生物科技有限公司 Soluble hydrogel microsphere and preparation method thereof and the application in Single cell analysis
CN113814008A (en) * 2020-08-28 2021-12-21 上海交通大学 Microfluidic channel structure, chip, particle ordered arrangement method and application
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

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242060A1 (en) * 2021-05-15 2022-11-24 墨卓生物科技(浙江)有限公司 Microsphere with specific oligonucleotide sequence for single-cell sequencing
CN117496274A (en) * 2023-12-29 2024-02-02 墨卓生物科技(浙江)有限公司 Classification counting method, system and storage medium based on liquid drop images
CN117496274B (en) * 2023-12-29 2024-06-11 墨卓生物科技(浙江)有限公司 Classification counting method, system and storage medium based on liquid drop images

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Application publication date: 20210806