A nucleic acid-labeled coded microsphere and its preparation method and application

Abstract

The present invention discloses a nucleic acid-labeled coding microsphere and a preparation method and application thereof. The preparation method uses a biological enzymatic reaction to synthesize nucleic acid labeling fragments, and realizes in situ synthesis of different oligonucleotides through the catalysis of biological enzymes, thereby providing sufficient storage capacity for encoding sequencing information. Compared with chemical synthesis, this method does not require complex steps such as segmented synthesis, connection, and assembly in traditional methods, which not only improves the efficiency and yield of the synthesis process, reduces production costs, but also enhances the specificity and stability of nucleic acid labeling. At the same time, the use of biological enzymes allows the reaction process to be carried out under mild conditions, which not only reduces the occurrence of side reactions, can achieve efficient synthesis in a shorter time, but also reduces dependence on chemical reagents, reduces the risk of environmental pollution and the risk of using harmful chemical reagents, thereby making the synthesis process of barcodes more sustainable and safe.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nucleic acid-labeled coding microsphere and a preparation method and application thereof. Background Art

[0002] The development of life science is a process of continuous refinement from macro to micro. Leeuwenhoek's improvement of the microscope in the 17th century opened the door to the cell era. In 1953, Watson and Crick determined the double helix structure of DNA and outlined the process of genetic information transmission. In 1977, Gilbert's invention of the Chemistry Prize and Sanger's invention of the chain termination sequencing method made it possible to determine the nucleic acid sequence. Today, many life science-related studies revolve around cells and nucleic acid sequences. The rise of high-throughput sequencing technology makes it possible to perform large-scale parallel sequencing of a large number of nucleic acid molecules.

[0003] Many times, researchers may not necessarily need to focus on the entire organ, but a smaller local tissue. Traditional bulk sequencing methods usually mix the RNA of thousands of cells together for analysis, thus masking the heterogeneity between cells. Single-cell sequencing separates individual cells from tissues or cell groups and performs separate sequencing analysis on the gene expression of each cell. It allows scientists to study gene expression and other genomic characteristics at the single cell level. The development of single-cell sequencing technology has greatly promoted our understanding of cell heterogeneity, cell state, and cell function. Spatial Multiomics is an emerging technology that can detect the expression of the entire transcriptome in cells while retaining the fine spatial positioning of cells. It is crucial for studying cell subpopulations and their molecular mechanisms that play a key role in the development or development of diseases.

[0004] Encoded microspheres contain structures such as barcode sequences, unique molecular identifiers (UMIs), and capture sequences (such as polyT), and play an important role in the application of single-cell sequencing and spatial omics. In single-cell sequencing, encoded microspheres can specifically bind to individual cells and mark cells through the barcode sequences on the microspheres to distinguish different cells. In spatial omics sequencing, encoded microspheres retain the location information of mRNA molecules through barcode sequences.

[0005] In the conventional production process of coded microspheres, nucleic acid barcodes are usually divided into multiple fragments for synthesis. These nucleic acid fragments are generally synthesized step by step using chemical synthesis methods, such as solid-phase synthesis, to form short-chain nucleic acid sequences. To ensure the quality and specificity of each fragment, each fragment synthesized in this process needs to be purified, which is time-consuming and labor-intensive. After the synthesis is completed, these nucleic acid fragments will not be used directly for coding microspheres, but need to be further subjected to complex splicing and assembly steps to obtain a complete barcode sequence with sufficient library capacity. Finally, the spliced ​​nucleic acid barcodes need to be connected to the surface of the microspheres for subsequent analysis and determination. This process involves multiple steps, including the synthesis of nucleic acid fragments, Purification, and subsequent connection and assembly steps. In this process, the synthesized nucleic acid fragments may be affected by different chemical reagents and reaction conditions, resulting in impurities or lower than expected yields. In addition, this method has high requirements for the laboratory environment during operation, and the process is complex, which increases process control and material costs. It can be seen that the current nucleic acid-labeled coded microspheres have many problems such as high production cost, difficulty in preparation, low preparation success rate, instability between different batches, etc., which seriously affect the accuracy and stability of sequencing results. At the same time, it also makes single-cell sequencing expensive and cannot be widely used. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing coding microspheres for synthesizing oligonucleotide sequences in situ on the microspheres through biological enzymatic reactions and its application.

[0007] In order to achieve the above object, the present invention first provides a method for preparing nucleic acid-labeled coded microspheres.

[0008] The method for preparing nucleic acid-labeled coding microspheres provided by the present invention comprises the following steps:

[0009] 1) fixing segment 1 to the microsphere to obtain the microsphere with fixed segment 1;

[0010] The segment 1 is a single-stranded DNA with a length of 4-100 bp;

[0011] 2) After completing step 1), the microspheres in the fixed section 1 are evenly divided into four reaction systems, and enzymatic DNA synthesis reaction system 1, enzymatic DNA synthesis reaction system 2, enzymatic DNA synthesis reaction system 3 and enzymatic DNA synthesis reaction system 4 are added respectively to carry out enzymatic reaction, centrifuge after the reaction is completed, discard the supernatant, and collect the microsphere precipitate; all the microsphere precipitates are mixed and resuspended in a buffer to obtain a resuspension;

[0012] The enzymatic DNA synthesis reaction system 1 includes a buffer, 3-ONH 2 -dATP and TdT enzyme;

[0013] The enzymatic DNA synthesis reaction system 2 includes a buffer, 3-ONH 2 -dTTP and TdT enzymes;

[0014] The enzymatic DNA synthesis reaction system 3 includes a buffer, 3-ONH 2 -dGTP and TdT enzymes;

[0015] The enzymatic DNA synthesis reaction system 4 includes a buffer, 3-ONH 2 -dCTP and TdT enzymes;

[0016] 3) After completing step 2), repeating step 2) at least 4 times with the resuspension to obtain microspheres connecting segment 1 and segment 2;

[0017] 4) After completing step 3), centrifuging the microspheres of the connecting section 1 and section 2, and discarding the supernatant; adding the enzymatic DNA synthesis reaction system 5 to carry out the enzymatic reaction, centrifuging after the reaction, discarding the supernatant, and collecting the microsphere precipitate; adding a buffer to the microsphere precipitate to resuspend it to obtain a resuspension; the enzymatic DNA synthesis reaction system 5 includes a buffer, 3-ONH 2 -dATP, 3-ONH 2 -dTTP, 3-ONH 2 -dGTP, 3-ONH 2 -dCTP and TdT enzymes;

[0018] 5) After completing step 4), repeating step 4) at least 8 times with the resuspension to obtain microspheres connecting segment 1, segment 2 and segment 3;

[0019] 6) After completing step 5), the microspheres connecting segment 1, segment 2 and segment 3 are centrifuged and the supernatant is discarded; a reaction system is added to react to obtain microspheres connecting segment 1, segment 2, segment 3 and segment 4; the reaction system includes a buffer, dATP and TdT enzyme.

[0020] In the above method, the nucleic acid marker can be single-stranded DNA or single-stranded RNA. Specifically, the nucleic acid marker is single-stranded DNA.

[0021] The structure of the nucleic acid marker includes but is not limited to the following segments: segment 1, segment 2, segment 3 and segment 4.

[0022] The sequence of segment 1 is random and is a universal sequence, and its 5' end is chemically modified, such as carboxyl modification, amino modification or thiol modification, for covalent binding with the microsphere.

[0023] Preferably, segment 1 is the entire or partial sequence of the illumina sequencing primer Read1 or Read2.

[0024] In some embodiments, the nucleotide sequence of segment 1 is as shown in sequence 1 in the sequence listing.

[0025] In some specific embodiments, the segment 1 may further include a dI modification between the deoxyribonucleotide at position 7 and the deoxyribonucleotide at position 8.

[0026] In some embodiments, the 5' end of segment 1 may also include a DBCO modification.

[0027] The total length of segment 2 and segment 3 is 12-80 bp.

[0028] The length of the segment 2 is preferably 4-16 bp. The segment 2 is a barcode sequence on the microsphere, and the barcode sequence on each microsphere is unique, which can mark the source of mRNA, thereby distinguishing different cells or samples.

[0029] The length of segment 3 is preferably 8-16 bp. In single-cell sequencing, segment 3 is usually used together with segment 2, wherein segment 2 is used to identify different cells or samples, and segment 3 is used to identify and quantify RNA molecules.

[0030] The segment 4 is a polyT tail with a length of 10-60 bp, which is used to specifically capture mRNA molecules in cells.

[0031] In the above method, in step 1), the particle size of the microspheres may be 1-300 µm.

[0032] In some embodiments, the microspheres have a particle size of 50 μm.

[0033] In the above method, in step 1), the microsphere can be connected to the nucleic acid marker via a breakable chemical bond. The breakable chemical bond includes but is not limited to a covalent bond (such as a nitrogen-nitrogen bond, an amide bond, etc.), a van der Waals force, an electrostatic interaction, a hydrogen bond, and a coordination bond.

[0034] The microspheres are selected from any one of the following: ferroferric oxide magnetic microspheres, carboxyl magnetic beads, carboxyl polystyrene microspheres, silica microspheres, N-hydroxysuccinimide microspheres, carboxyl microspheres, agarose gel microspheres, polyacrylamide microspheres, polyethylene glycol microspheres, hard microspheres, hydrogel microspheres, hard and soft mixed microspheres, magnetic microspheres and non-magnetic microspheres.

[0035] In some embodiments, the microspheres are azide-modified microspheres. The azide-modified microspheres are synthesized by using acrylamide (AW), ammonium persulfate (APS), N,N-methylenebisacrylamide (MBAM), N,N,N',N'-tetramethylethylenediamine, and 2-methacryloyloxyethyltrimethylammonium chloride through a microfluidic device and a chip, and the specific preparation method thereof may include the following steps:

[0036] a. Prepare aqueous solution

[0037] Mixing water with acrylamide, an initiator ammonium persulfate, a crosslinking agent N,N-methylenebisacrylamide, a reaction accelerator N,N,N',N'-tetramethylethylenediamine, and 2-methacryloyloxyethyltrimethylammonium chloride to obtain an aqueous phase solution;

[0038] b. Prepare oil phase solution

[0039] Mixing liquid paraffin with cetyl polyethylene glycol ABIL EM90, sorbitan oleate and Tween 80 to obtain an oil phase solution;

[0040] c. Connect the microfluidic device and three syringe pumps equipped with syringes to three polytetrafluoroethylene tubes respectively, wherein two conduits flow the oil phase solution, and the other conduit flows the water phase solution. The outlet of the microfluidic device flows through a polytetrafluoroethylene extension tube into a 95-degree oil bath. Under the action of shear force, the water phase liquid is sheared into evenly dispersed droplets and solidified into spheres after passing through a water bath.

[0041] In the above method, in step 1), the method of fixing segment 1 to the microsphere may include the following steps:

[0042] 1-1) Centrifuge the microsphere solution, discard the supernatant, and collect the precipitate;

[0043] 1-2) adding buffer and segment 1 to the precipitate, mixing, reacting, centrifuging, discarding the supernatant, and collecting the precipitate;

[0044] 1-3) adding a buffer to the precipitate, centrifuging, discarding the supernatant, and collecting the precipitate;

[0045] 1-4) Repeat steps 1-3);

[0046] 1-5) Add buffer to the precipitate to obtain microspheres connected to segment 1.

[0047] Furthermore,

[0048] In the above 1-1), the microsphere solution may be an azide-modified microsphere solution with a concentration of 10 mg / mL.

[0049] The centrifugal condition may be centrifugation at 10,000 rpm for 1 min.

[0050] In 1-2), the buffer may be 20X UltraPure™ SSC Buffer.

[0051] The reaction conditions may be shaking at 900 rpm and 30° C. for 30 min.

[0052] The centrifugal condition may be centrifugation at 10000 rpm for 30 s.

[0053] In 1-3), the buffer may be a phosphate buffer.

[0054] The centrifugal condition may be centrifugation at 10,000 rpm for 1 min.

[0055] In the above 1-5), the buffer may be a phosphate buffer.

[0056] In the above method, in step 2), the step of dividing the reaction system into four reaction systems may further include a centrifugation step. The centrifugation condition may be centrifugation at 10,000 rpm for 1 min.

[0057] The buffer in the enzymatic DNA synthesis reaction system can be phosphate buffer.

[0058] The enzymatic reaction may be carried out under shaking at 40° C. for 1 min.

[0059] The centrifugation condition after the enzymatic reaction may be centrifugation at 10,000 rpm for 30 s.

[0060] The method further includes the steps of deprotection and washing before mixing all microsphere precipitations and adding buffer solution for resuspending.

[0061] The deprotection solution used in the deprotection can be NaNO with a concentration of 0.7 M. 2 Solution.

[0062] The deprotection condition may be shaking at 40° C. for 30 s.

[0063] The deprotection step further includes a centrifugation step; the centrifugation condition may be centrifugation at 10000 rpm for 30 s.

[0064] The cleaning solution used in the cleaning may be phosphate buffer.

[0065] The cleaning may be performed twice.

[0066] The step of centrifugation is further included after the washing; the centrifugation condition may be centrifugation at 10,000 rpm for 30 s.

[0067] The buffer added after the washing may be a phosphate buffer.

[0068] In the above method, in step 3), the number of times step 2) is repeated can be 4-16 times. The number of times step 2) is repeated is the length of segment 2 added to the microsphere. If step 2) is repeated 4 times, the length of segment 2 added to the microsphere is 4 bp; if step 2) is repeated 8 times, the length of segment 2 added to the microsphere is 8 bp; if step 2) is repeated 12 times, the length of segment 2 added to the microsphere is 12 bp; if step 2) is repeated 16 times, the length of segment 2 added to the microsphere is 16 bp. In practical applications, the appropriate number of repetitions can be selected according to actual needs to add segment 2 of appropriate length.

[0069] In some embodiments, the step 4) is repeated 4 times, 8 times, 12 times or 16 times.

[0070] In the above method, in step 4), the centrifugation condition may be 10,000 rpm for 1 min.

[0071] The enzymatic reaction may be carried out under shaking at 40° C. for 1 min.

[0072] The centrifugation condition after the enzymatic reaction may be centrifugation at 10,000 rpm for 30 s.

[0073] The buffer in the enzymatic DNA synthesis reaction system 5 can be a phosphate buffer.

[0074] The 3-ONH 2 -dATP, 3-ONH 2 -dTTP, 3-ONH 2 -dGTP, 3-ONH 2 The molar ratio of -dCTP may be 1:1:1:1.

[0075] The method also includes deprotection and washing steps before adding buffer to the microsphere precipitate for re-suspending.

[0076] The deprotection solution used in the deprotection can be NaNO with a concentration of 0.7 M. 2 Solution.

[0077] The deprotection condition may be shaking at 40° C. for 30 s.

[0078] The deprotection step further includes a centrifugation step; the centrifugation condition may be centrifugation at 10000 rpm for 30 s.

[0079] The cleaning solution used in the cleaning may be phosphate buffer.

[0080] The cleaning may be performed twice.

[0081] The step of centrifugation is further included after the washing; the centrifugation condition may be centrifugation at 10,000 rpm for 30 s.

[0082] The buffer added after the washing may be a phosphate buffer.

[0083] In the above method, in step 5), the number of times step 4) is repeated can be 8-16 times. The number of times step 4) is repeated is the length of segment 3 added to the microsphere. If step 4) is repeated 8 times, the length of segment 3 added to the microsphere is 8 bp; if step 4) is repeated 12 times, the length of segment 3 added to the microsphere is 12 bp; if step 4) is repeated 16 times, the length of segment 3 added to the microsphere is 16 bp. In practical applications, the appropriate number of repetitions can be selected according to actual needs to add segment 3 of appropriate length.

[0084] In some embodiments, the step 4) is repeated 8 times.

[0085] In the above method, in step 6), the centrifugation condition may be 10,000 rpm for 1 min.

[0086] The reaction conditions may be shaking reaction at 30° C. for 5 min.

[0087] The buffer in the reaction system may be 10×PBS.

[0088] The solvent of any of the above-mentioned phosphate buffers is water, and the solute and its concentration are 50 mM KH 2 PO 4 , 100 mMNaCl, 0.06% Triton.

[0089] The nucleic acid-labeled coded microspheres prepared according to any of the above methods also fall within the protection scope of the present invention.

[0090] In order to achieve the above object, the present invention also provides a new use of the above nucleic acid labeled coding microspheres.

[0091] The present invention provides the use of the above-mentioned nucleic acid-labeled coding microspheres in single-cell sequencing or spatial omics sequencing.

[0092] The present invention also provides the use of nucleic acid-labeled coding microspheres in preparing products for single-cell sequencing or spatial omics sequencing.

[0093] In order to achieve the above objectives, the present invention finally provides a method for single-cell sequencing or spatial omics sequencing.

[0094] The encoding microspheres used in the single-cell sequencing or spatial omics sequencing methods provided by the present invention are the encoding microspheres labeled with the above-mentioned nucleic acid.

[0095] The present invention provides a method for preparing nucleic acid-labeled coding microspheres, which uses a biological enzymatic reaction to synthesize nucleic acid labeling fragments, and realizes in situ synthesis of different oligonucleotides (oligo) by catalysis of biological enzymes, thereby providing sufficient storage capacity for encoding sequencing information. Compared with chemical synthesis, this method does not require complex steps such as segmented synthesis, connection, and assembly in traditional methods, which not only improves the efficiency and yield of the synthesis process, reduces production costs, but also enhances the specificity and stability of nucleic acid labels. At the same time, the use of biological enzymes allows the reaction process to be carried out under mild conditions, which not only reduces the occurrence of side reactions, can achieve efficient synthesis in a shorter time, but also reduces dependence on chemical reagents, reduces the risk of environmental pollution and the risk of using harmful chemical reagents, thereby making the synthesis process of barcodes more sustainable and safe. Therefore, the preparation method of nucleic acid-labeled coding microspheres provided by the present invention can not only significantly improve the storage capacity of sequencing microspheres, but also has the advantages of simple process flow, low cost, low error rate, small batch-to-batch difference, and high safety. Based on the preparation method of the present invention, coding microspheres for single-cell sequencing and spatial omics sequencing can be mass-produced, promoting the development of single-cell sequencing and other genomics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 It is a schematic diagram of the structure of the "coded microsphere" of the present invention.

[0097] Figure 2 It is a schematic diagram of the process of preparing the "coded microspheres" of the present invention.

[0098] Figure 3 It is TdT enzymatic synthesis of oligo. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0100] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0101] Example 1. Preparation method of nucleic acid labeled coded microspheres

[0102] The schematic diagram of the structure of the nucleic acid-labeled coding microsphere of the present invention is as follows Figure 1 As shown. The microsphere material includes but is not limited to ferroferric oxide magnetic microspheres, carboxyl magnetic beads, carboxyl polystyrene microspheres, silica microspheres, N-hydroxysuccinimide microspheres, carboxyl microspheres, agarose gel microspheres, polyacrylamide microspheres, polyethylene glycol microspheres, hard microspheres, hydrogel microspheres, hard and soft mixed microspheres, magnetic microspheres or non-magnetic microspheres, etc. The diameter of the microspheres can be 1-300µm. The microspheres are connected to the nucleic acid fragments through breakable chemical bonds. Breakable chemical bonds include but are not limited to nitrogen-nitrogen bonds, amide bonds and other covalent bonds, van der Waals forces, electrostatic interactions, hydrogen bonds, coordination bonds, etc.

[0103] The modified nucleic acid marker on the microsphere can be a single-stranded ribonucleic acid (RNA) sequence or a single-stranded deoxyribonucleic acid (DNA) sequence, and its structure includes but is not limited to the following segments: segment 1, segment 2, segment 3 and segment 4. The length of segment 1 is 4-100 bp, which is a universal sequence with a random sequence, and its 5' end is chemically modified, such as carboxyl modification, amino modification or thiol modification, for covalent binding to the microsphere. Preferably, segment 1 is the entire or partial sequence of illumina sequencing primer Read1 or Read2. Segment 2 is a barcode sequence on the microsphere, preferably 4-16 bp in length. The barcode sequence on each microsphere is unique and can mark the source of mRNA, thereby distinguishing different cells or samples. The length of segment 3 is preferably 8-16 bp. In single-cell sequencing, segment 3 is usually used together with segment 2. Segment 2 is used to identify different cells or samples, while segment 3 is used to identify and quantify RNA molecules. Segment 4 is a polyT tail with a length of 10-60 bp, which is used to specifically capture mRNA molecules in cells.

[0104] The flowchart of the method for preparing nucleic acid-labeled encoded microspheres provided by the present invention is as follows: Figure 2 The present invention takes 50 μm azide-modified microspheres as a solid phase carrier as an example, and uses biological enzymes to synthesize the elements of segment 2, segment 3, and segment 4. The specific steps are as follows:

[0105] 1. Fixation of the probe (segment 1)

[0106] 1. Take 1000 µL of azide-modified microsphere solution with a final concentration of 10 mg / mL into a low-adsorption EP tube, centrifuge at 10000 rpm for 1 min, and discard the supernatant.

[0107] The above-mentioned azide-modified microspheres are synthesized by using acrylamide, ammonium persulfate, N,N-methylenebisacrylamide (MBAM), N,N,N',N'-tetramethylethylenediamine, and 2-methacryloyloxyethyltrimethylammonium chloride through a microfluidic device and a chip. The specific preparation method is as follows:

[0108] 1) Prepare 10 g of aqueous solution

[0109] 0.5 g acrylamide (AW), 0.01 g initiator ammonium persulfate (APS), 0.01 g cross-linking agent N,N-methylenebisacrylamide (MBAM), 0.01 g reaction accelerator N,N,N',N'-tetramethylethylenediamine, and 0.01 g 2-methacryloyloxyethyltrimethylammonium chloride were added to 9.46 g water and mixed to obtain 10 g aqueous phase solution.

[0110] 2) Prepare 100 g oil phase solution

[0111] Liquid paraffin was selected as the continuous phase, and 0.5 g of cetyl polyethylene glycol ABILEM90, 2 g of sorbitan oleate (Span80) and 1 g of Tween 80 were added to 96.5 g of liquid paraffin to obtain 100 g of an oil phase solution.

[0112] 3) The microfluidic device was connected to three syringe pumps equipped with syringes and three polytetrafluoroethylene tubes, two of which were used to circulate the oil phase solution and the other one to circulate the water phase solution. The water phase flow rate was 2 mL / h and the oil phase flow rate was 15 mL / h. The outlet of the microfluidic device flowed through a polytetrafluoroethylene extension tube into a 95-degree oil bath. Under the action of shear force, the water phase liquid was sheared into uniformly dispersed droplets and solidified into spheres after passing through a water bath. Then it flowed through the tube into a beaker filled with cyclohexane / water (the volume ratio of cyclohexane to water was 3:1) to form microspheres with a particle size of 50 µm.

[0113] 2. Add the reaction system shown in Table 1 below to the EP tube in step 1, mix by pipetting, and oscillate at 900 rpm and 30°C for 30 min.

[0114]

[0115] Note: ide oxy i indicates (deoxyhypoxanthine, di) modification.

[0116] 3. Centrifuge at 10,000 rpm for 30 s and discard the supernatant.

[0117] 4. Add 400 µL of phosphate buffer (50 mM KH 2 PO 4, 100 mM NaCl, 0.06% Triton, pH 6.8), centrifuge at 10,000 rpm for 1 min, and discard the supernatant.

[0118] 5. Repeat step 4.

[0119] 6. Add 800 µL of phosphate buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 0.06% Triton, pH 6.8) to dissolve back to obtain microspheres connected to segment 1 for later use.

[0120] 2. Enzymatic reaction synthesis section 2

[0121] 1. Mix 800 µL of the microspheres of connection segment 1 obtained in step 1 and divide them into 4 reaction systems, each containing 200 µL of the microspheres of connection segment 1. Centrifuge each reaction system at 10,000 rpm for 1 min and discard the supernatant.

[0122] 2. Add the enzymatic DNA synthesis reaction system shown in Table 2 to the four EP tubes in step 1 respectively, wherein each reaction system uses a different nucleotide substrate.

[0123]

[0124] Note: 3-ONH 2 -dATP, 3-ONH 2 -dTTP, 3-ONH 2 -dGTP, 3-ONH 2 -dCTP Correspondence Figure 2 3-O-am-dA, 3-O-am-dG, 3-O-am-dT and 3-O-am-dC in Step 3.

[0125] 3. Oscillate the reaction at 40°C for 1 min, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0126] 4. Put the four tubes of microspheres into one tube and add deprotection solution (0.7M NaNO 2 ) 500 μL, shake at 40℃ for 30 s for deprotection, centrifuge at 10000 rpm for 30 s, and discard the supernatant.

[0127] 5. Add 200 µL of phosphate buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 0.06% Triton, pH 6.8) and mix well, wash by pipetting, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0128] 6. Repeat step 5.

[0129] 7. Add 200 μL phosphate buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 0.06% Triton, pH 6.8).

[0130] 8. Repeat steps 1-7 16 times to obtain microspheres connecting segment 1 and segment 2.

[0131] 3. Enzymatic reaction synthesis section 3

[0132] 1. Mix the microspheres of connection section 2 obtained in step 2 respectively, centrifuge at 10000 rpm for 1 min, and discard the supernatant.

[0133] 2. Add the enzymatic DNA synthesis reaction system shown in Table 3 to the EP tube in step 1, wherein the substrate is a uniform mixture of four nucleotides.

[0134]

[0135] Note: 3-ONH 2 -dATP, 3-ONH 2 -dTTP, 3-ONH 2 -dGTP, 3-ONH 2 -dCTP Correspondence Figure 2 3-O-am-dA, 3-O-am-dG, 3-O-am-dT and 3-O-am-dC, 3-ONH in Step 6 2 -dATP, 3-ONH 2 -dTTP, 3-ONH 2 -dGTP, 3-ONH 2 The molar ratio of -dCTP is 1:1:1:1.

[0136] 3. Oscillate the reaction at 40°C for 1 min, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0137] 4. Add 500 µL of deprotection solution, shake at 40°C for 30 s, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0138] 5. Add 200 µL phosphate buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 0.06% Triton, pH 6.8) and mix well, wash by pipetting, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0139] 6. Repeat step 5.

[0140] 7. Add 800 µL phosphate buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 0.06% Triton, pH 6.8).

[0141] 8. Repeat steps 1-7 8 times to obtain microspheres connecting segment 1, segment 2 and segment 3.

[0142] 4. Add Section 4

[0143] 1. Mix the microspheres of connected segments 1, 2 and 3 obtained in step 3, centrifuge at 10,000 rpm for 1 min, and discard the supernatant.

[0144] 2. Add the reaction system shown in Table 4 to the EP tube in step 1, and shake at 30°C for 5 min to obtain microspheres connecting segment 1, segment 2, segment 3, and segment 4.

[0145]

[0146] 5. Chain cutting

[0147] 1. Mix the microspheres of connected segments 1, 2, 3 and 4 obtained in step 4, centrifuge at 10,000 rpm for 1 min, and discard the supernatant.

[0148] 2. Add the reaction system shown in Table 5 to the EP tube in step 1, perform enzyme digestion at 65°C for 30 min, and react at 98°C for 2 min to inactivate the enzyme and obtain the reaction product.

[0149]

[0150] 6. Electrophoresis

[0151] 1. Sample: Take 10 µL of the reaction product obtained in step 5 and add 10 µL of loading buffer for electrophoresis.

[0152] 2. Gel preparation: Gel preparation was performed according to the following formula: 16 mL 20% urea gel preparation solution, 100 µL 10% APS, and 8 µL TEMED.

[0153] 3. Gel running: Load 10 µL sample and run at 450V for 120 min.

[0154] 4. Gel staining: Add 12 µL SYBR TM Gold dye was added to 120 mL of 1×TBE and shaken at 70 rpm for 15 min.

[0155] 5. Glue.

[0156] The results are as follows Figure 3 As shown, lane 1 is a DNA Marker, lane 2 is a negative control without step 2, N+4 in lanes 3 and 4 indicates the product of adding 4 bp in step 2, N+8 in lanes 5 and 6 indicates the product of adding 8 bp in step 2, N+12 in lanes 7 and 8 indicates the product of adding 12 bp in step 2, and N+16 in lanes 9 and 10 indicates the product of adding 16 bp in step 2. It can be seen from the figure that compared with the negative control group, the product of the N+4 group has 4 more bases, the product of the N+8 group has 8 more bases, the product of the N+12 group has 12 more bases, and the product of the N+16 group has 16 more bases.

[0157] Example 2: Performance testing of nucleic acid-labeled coded microspheres

[0158] In this example, the single cell transcriptome detection kit of 10X Genomics (kit name is Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1, 10X Genomics, catalog number: CG000315 Rev F) was used as a control to evaluate the performance of the nucleic acid-labeled encoding microspheres prepared in Example 1. The specific steps are as follows:

[0159] 1. Cell Culture

[0160] The human erythroleukemia cell line K562 was revived and cultured in a 6 cm diameter culture dish in a high glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (PS).

[0161] 2. Single Cell Labeling

[0162] 1. Prepare a single cell reaction solution according to the reaction system shown in Table 6.

[0163]

[0164] Note: The components in Table 6 are all from the Chromium Next GEM Single Cell 3ʹ Reagent Kitsv3.1 kit.

[0165] 2. Prepare the experimental chip (from Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1) and add 80 µL (1×10 5 / mL) K562 cell solution, and add single cell reaction solution after the cells cover the chip. The single cell reaction solution consists of 40 μL encoding microspheres / 10×genomics beads and 280 μL Preparation Oil. Among them, the encoding microspheres are nucleic acid-labeled encoding microspheres prepared in Example 1 (microspheres with 16 bp segment 2 added), and the 10×genomics beads come from the Chromium Next GEM Single Cell 3ʹ ReagentKits v3.1 kit.

[0166] 3. Place the chip into a cell labeling instrument to generate droplets, lyse cells, and capture mRNA at room temperature.

[0167] 4. After the reaction is completed, remove the chip and take 125 µL of the reaction product into a PCR tube.

[0168] 3. cDNA Purification

[0169] 1. Incubate the reaction product obtained in step 2 according to the procedure shown in Table 7 (heat cover temperature 53°C).

[0170]

[0171] 2. Add 125 µL Recovery Agent to the product obtained in step 1 and let stand for 2 min.

[0172] 3. Slowly aspirate 125 µL of product from the bottom of the tube, and do not aspirate the supernatant.

[0173] 4. Prepare the purified solution according to the reaction system shown in Table 8.

[0174]

[0175] Note: The components in Table 8 are all from the Chromium Next GEM Single Cell 3ʹ Reagent Kitsv3.1 kit.

[0176] 5. Add 200 µL of purification solution to each sample, vortex to mix, and incubate at room temperature for 10 min.

[0177] 6. After incubation for 10 min, place it on a magnetic rack until the solution becomes clear and discard the supernatant.

[0178] 7. Add 300 µL of 80% ethanol, wait for 30 seconds, and discard the supernatant.

[0179] 8. Repeat step 7 above.

[0180] 9. After a brief centrifugation, let stand at room temperature for 1 min.

[0181] 10. Immediately add 35.5 µL of the eluent shown in Table 9.

[0182]

[0183] Note: The components in Table 9 are all from the Chromium Next GEM Single Cell 3ʹ Reagent Kitsv3.1 kit.

[0184] 11. After standing at room temperature for 2 min, place on a magnetic rack and transfer the supernatant to a new EP tube.

[0185] IV. cDNA Amplification

[0186] 1. Prepare the amplification system according to the formula shown in Table 10.

[0187]

[0188] Note: The components in Table 10 are all from the Chromium Next GEM Single Cell 3ʹ Reagent Kitsv3.1 kit.

[0189] 2. Perform amplification according to the amplification program shown in Table 11.

[0190]

[0191] 3. Vortex to resuspend SPRIselect reagent. Add 60 µL SPRIselect reagent (0.6X) to each sample and incubate at room temperature for 5 min.

[0192] 4. Place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0193] 5. Add 300 µL of 80% ethanol, wait for 30 s, and discard the supernatant.

[0194] 6. Repeat step 5 above.

[0195] 7. After a brief centrifugation, let stand at room temperature for 1 min.

[0196] 8. Immediately add 40 µL of elution buffer EB, let stand at room temperature for 2 min, and take the supernatant into a new EP tube.

[0197] 5. Library Construction

[0198] The library was constructed according to the 3ʹ Gene Expression Dual Index Library Construction in the 10X Genomics single-cell transcriptome detection kit.

[0199] VI. Library Quality Inspection

[0200] The library concentration was measured using Qubit.

[0201] The results are shown in Table 12. The control group refers to the data obtained using the microspheres of 10× Company, and the test group refers to the data obtained using the nucleic acid-labeled encoded microspheres prepared in Example 1.

[0202]

[0203] VII. Sequencing and Analysis

[0204] The Illumina NovaSeq 6000 sequencer was used for sequencing to analyze parameters such as the number of effective cells captured by the encoded microspheres, the number of molecular tag UMIs, and the number of captured genes.

[0205] The results are shown in Table 13, which show that the coded microspheres produced by the present invention can achieve the same effect as the products of 10×Genomics in single-cell analysis. At present, 10×Genomics uses a multi-step connection method (CN111051523A) to prepare microspheres. Since the reaction requires multi-step connection, and the coded microspheres generated by the connection reaction have only 80% of the final product after 3-fold connection, and each step requires purification and recovery, resulting in a 3-step connection purification recovery rate of about 50%, and half of the reagent loss. In addition, this method requires the synthesis of a large number of DNA sequences with special modifications, resulting in high costs and long experimental cycles. The homemade coded microspheres of the present invention do not need to synthesize a large number of DNA sequences or perform multiple connections. The DNA sequence is synthesized on the stationary phase by an enzymatic method, and the enzymatic synthesis only takes 1 min to achieve the effect of 10×Genomics' products, with a short experimental cycle, low cost, and more marketability.

[0206] .

Claims

1. A method for preparing nucleic acid-labeled encoded microspheres, comprising the following steps: 1) fixing segment 1 to the microsphere to obtain the microsphere with fixed segment 1; The segment 1 is a single-stranded DNA with a length of 4-100 bp; 2) After completing step 1), the microspheres in the fixed section 1 are evenly divided into four reaction systems, and enzymatic DNA synthesis reaction system 1, enzymatic DNA synthesis reaction system 2, enzymatic DNA synthesis reaction system 3 and enzymatic DNA synthesis reaction system 4 are added respectively to carry out enzymatic reaction, centrifuge after the reaction is completed, discard the supernatant, and collect the microsphere precipitate; all the microsphere precipitates are mixed and deprotected; after the deprotection is completed, a buffer solution is added for re-suspending to obtain a resuspension; The enzymatic DNA synthesis reaction system 1 is composed of a buffer, 3-ONH2-dATP and TdT enzyme; The enzymatic DNA synthesis reaction system 2 is composed of a buffer, 3-ONH2-dTTP and TdT enzyme; The enzymatic DNA synthesis reaction system 3 is composed of a buffer, 3-ONH2-dGTP and TdT enzyme; The enzymatic DNA synthesis reaction system 4 is composed of a buffer, 3-ONH2-dCTP and TdT enzyme; 3) After completing step 2), repeating step 2) at least 4 times with the resuspension to obtain microspheres connecting segment 1 and segment 2; 4) After completing step 3), centrifuging the microspheres of the connecting section 1 and section 2, and discarding the supernatant; adding the enzymatic DNA synthesis reaction system 5 to carry out the enzymatic reaction, centrifuging after the reaction, discarding the supernatant, and collecting the microsphere precipitate; deprotecting the microsphere precipitate, and adding a buffer to resuspend after the deprotection is completed to obtain a resuspension; the enzymatic DNA synthesis reaction system 5 is composed of a buffer, 3-ONH2-dATP, 3-ONH2-dTTP, 3-ONH2-dGTP, 3-ONH2-dCTP and TdT enzyme; 5) After completing step 4), repeating step 4) at least 8 times with the resuspension to obtain microspheres connecting segment 1, segment 2 and segment 3; 6) After completing step 5), the microspheres connecting segment 1, segment 2 and segment 3 are centrifuged and the supernatant is discarded; a reaction system consisting of a buffer, dATP and TdT enzyme is added to react to obtain microspheres connecting segment 1, segment 2, segment 3 and segment 4; The nucleic acid marker is single-stranded DNA; The segment 1 is a universal sequence, the 5' end of which is chemically modified for covalent binding to the microspheres; The length of segment 2 is 4-16 bp; The length of segment 3 is 8-16 bp; The segment 4 is a polyT tail with a length of 10-60 bp; The particle size of the microspheres is 1-300 μm.

2. The method according to claim 1, characterized in that: In the step 1), the nucleotide sequence of segment 1 is shown in sequence 1.

3. The method according to claim 1, characterized in that: In the step 1), the microspheres are azide-modified microspheres.

4. The method according to claim 1, characterized in that: In the step 2), a washing step is also included after the deprotection.

5. The method according to claim 1, characterized in that: In the step 4), a washing step is also included after the deprotection.

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