Microbial single-cell transcriptome sequencing method based on hydrogel microspheres

Abstract

The present invention discloses a method for sequencing the transcriptome of a single microorganism cell based on hydrogel microspheres. This method relates to the field of single-cell sequencing and can implement a sequencing process with a relatively low number of microorganisms. This method provides the possibility of high-throughput single-cell sequencing for samples with relatively small numbers of microorganisms, thereby resolving the bias in results caused by microbial sample loss. The method comprises: fixing a microbial sample, preparing the fixed microbial sample into hydrogel microspheres, synthesizing cDNA from the RNA of the microbial cells within the microspheres, labeling the cDNA, and constructing and sequencing a library of the labeled cDNA to obtain transcriptome information for the single microbial cell. The microspheres are porous network structures, and the pores on their surfaces allow proteins and reagents to pass through, thereby blocking individual microorganisms within the microspheres.

Description

Technical Field

[0001] The present invention relates to the field of single-cell sequencing, and in particular to a microbial single-cell transcriptome sequencing method based on hydrogel microspheres. Background Art

[0002] Single-cell transcriptome sequencing (SCTS) technology can provide transcriptome information at single-nucleotide resolution within a single cell. This technology has become a popular tool for characterizing eukaryotic cell diversity, significantly improving our understanding of transcriptional heterogeneity across cell types and states. However, when applied to microbial cells, SCTS suffers from the challenges of aggregation and isolation due to their small size. Furthermore, factors such as the diverse cell walls of microorganisms and the lack of polyadenylation tails on mRNA (messenger deoxyribonucleic acid) significantly hinder its application.

[0003] Recently developed high-throughput single-cell RNA sequencing methods for bacteria, including microSPLiT (microbial split-pool ligation transcriptomics), PETRI-seq (Prokaryotic Expression Profiling by Tagging RNA In Situ and Sequencing), BacDrop (droplet-based whole-genome parallel large-scale bacterial single-cell RNA sequencing), and smRandom-seq (droplet microfluidics-based high-throughput single-microorganism RNA sequencing analysis), overcome the inherent limitations of microorganisms while significantly increasing sequencing throughput, providing a platform for the application of microbial single-cell RNA (ribonucleic acid) sequencing. However, due to the repeated use of centrifugation in these technical processes, individual microorganisms are easily lost, placing high demands on the test sample. To compensate for the errors caused by individual loss, an input sample amount of at least tens of millions is required. Furthermore, because microbiome samples contain a wide variety of microorganisms and their volumes vary at the species level, the loss of various microorganisms in the sample after centrifugation varies, often leading to significant bias in the final results. Furthermore, for some precious microbiome samples, the inherent loss of microorganisms due to the relatively small number of microorganisms in the sample makes them unsuitable for high-throughput single-bacteria RNA sequencing technologies.

[0004] In summary, the high-throughput single-cell sequencing methods in the existing technology have extremely high requirements for test samples, and also have the problems of severe centrifugal loss and large differences in centrifugal loss, which leads to serious deviations in sequencing results. Summary of the Invention

[0005] The present invention provides a microbial single-cell transcriptome sequencing method based on hydrogel microspheres, which can realize the sequencing process with less than 100,000 microorganisms, providing the possibility of high-throughput single-cell sequencing for samples with a small number of microorganisms, and can solve the result deviation caused by the loss of microbial samples, solving the problem of microbial loss in microbial single-cell sequencing technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Microbial single-cell transcriptome sequencing methods based on hydrogel microspheres include:

[0008] The microbial sample is fixed and prepared into hydrogel microspheres. The RNA of the microbial cells is synthesized into cDNA inside the microspheres, and the cDNA is labeled. The labeled cDNA is then library-built and sequenced to obtain the transcriptome information of the microbial single cell.

[0009] The microspheres have a porous network structure, and the pores on the surface can pass through proteins and reagents, thereby blocking the microorganisms inside the microspheres.

[0010] Furthermore, the sequencing method specifically comprises the following steps:

[0011] The tested microbial sample is prepared into a single cell suspension, and uniformly mixed with a fixative to obtain a fixed microbial sample;

[0012] mixing the fixed microbial sample and the hydrogel material to obtain a hydrogel solution containing the microbial sample;

[0013] The hydrogel solution containing the microbial sample is prepared into water-in-oil droplets, and the hydrogel material in the droplets is solidified;

[0014] The water-in-oil droplets are demulsified and washed to obtain microspheres encapsulating the microbial sample;

[0015] Digest and lyse the cell walls of the microbial samples in the microspheres, allowing the reagent to pass through the cell walls and enter the cells of the microbial samples;

[0016] The reverse transcription primer is used to perform an in situ reverse transcription reaction on the RNA of the microbial cell to synthesize the first strand of cDNA, and then the cDNA is labeled;

[0017] The microspheres are dissolved, the cDNA released from the microspheres is purified, and the purified cDNA is library-built and sequenced to obtain the transcriptome information of the microbial single cell.

[0018] Furthermore, the fixative is a simple fixative or a mixed fixative.

[0019] Simple fixatives include one of paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, and mercuric chloride.

[0020] Mixed fixatives include various types of acetic acid-alcohol mixtures, formalin-acetic acid-alcohol solutions, and Boyne's fixatives.

[0021] Furthermore, the hydrogel microspheres can be dissolved after solidification.

[0022] Furthermore, the materials used for the hydrogel microspheres include agarose, chitosan, gelatin, hyaluronic acid, polyacrylamide, polyvinyl alcohol, polyethylene glycol, poly (N-isopropylacrylamide), alginate, carrageenan, and polyacrylic acid.

[0023] Furthermore, the preparation method of the hydrogel solution is:

[0024] Mix 7.5ul of 40% acrylamide solution, 2.5ul of 5% N,N'-bis(acryloyl)cystamine solution, 2.5ul of 10% ammonium persulfate solution, and 15ul of PBS (Phosphate Buffered Saline), and then add ultrapure water to 50ul.

[0025] The microspheres obtained by preparing the hydrogel solution using this method have good permeability.

[0026] Furthermore, each microsphere contains no or only one microorganism.

[0027] Furthermore, the preparation method of the water-in-oil droplets includes:

[0028] In microfluidics, incompatible liquids are connected through microchannels to generate droplets at the intersection, which are then cross-linked to form hydrogel microspheres;

[0029] The emulsion method uses two immiscible solvents under the action of surfactants to form a uniform emulsion, and precipitates the solid phase from the emulsion, so that the nucleation, growth, aggregation and other processes are confined to small spherical droplets;

[0030] Batch emulsification, where incompatible liquids are mixed together to form cross-linkable hydrogel droplets;

[0031] 3D printing method;

[0032] Photolithography, which focuses light on a mask or mold to solidify and cross-link to form hydrogel microspheres;

[0033] The electrospray method is to apply a voltage between the needle and the receiving liquid so that the applied voltage overcomes the surface tension at the needle tip, forming a charged droplet jet that cross-links in the receiving liquid to form hydrogel microspheres.

[0034] Further, the method of cDNA labeling includes:

[0035] A barcoded reverse transcription primer is used to capture all RNA in the microbial sample, and the first chain of cDNA is synthesized in situ by reverse transcription on the RNA, and a first round of labeling is added at the same time, followed by one or more labels.

[0036] To add a tag once:

[0037] A poly(dA) tail is added to the 3' end of the cDNA in situ using terminal transferase, followed by further labeling using a barcoded primer and simultaneous second-strand cDNA synthesis.

[0038] To add multiple markers:

[0039] Two more rounds of barcode labeling are performed. Through ligase, the barcodes of the next two rounds will be connected to the barcode of the first round. Finally, the cDNA is purified and the second chain synthesis is performed.

[0040] Further, the method for labeling cDNA includes:

[0041] Poly(A) (polyadenosine) polymerase I is used to perform in situ polyadenylation on RNA (ribonucleic acid), and random hexamer primers with barcode labels and poly(dT) (polydeoxythymidine nucleotide) primers with barcode labels are used to perform the first round of labeling and reverse transcription reaction on microorganisms to synthesize the first chain of cDNA. The second and third rounds of barcode labeling are then performed using ligase, and finally the cDNA is purified and the second chain of cDNA is synthesized.

[0042] The beneficial effects of the present invention are:

[0043] The present invention is suitable for high-throughput single-bacteria analysis of microbial samples with relatively small starting amounts (capable of measuring information on thousands of bacteria with a minimum starting amount of 50,000). This addresses the issue of microbial loss in single-cell microbial sequencing, effectively reducing the required starting amount and avoiding the adhesion of individual microorganisms caused by multiple centrifugation. Furthermore, because encoding is performed simultaneously during cDNA synthesis, the cDNA is compatible with a variety of high-throughput single-cell microbial sequencing technologies, including those based on microfluidics. This method can also be used for high-throughput sequencing of small, precious microbial samples.

[0044] This method encapsulates individual microorganisms into hydrogel microspheres roughly the size of eukaryotic cells. These uniformly sized microspheres can be rapidly precipitated at 2000g for 1 minute, eliminating the need for further centrifugation based on bacterial species. Different microorganisms encapsulated in the same-sized microspheres will not experience different sedimentation rates due to differences in size and weight, thus minimizing variations in loss during centrifugation.

[0045] The microspheres encapsulating the microorganisms in this invention have a porous network structure that allows proteins and various reagents to pass through, but is not porous enough for individual microorganisms to pass through. This allows for direct RNA capture and linker addition to the encapsulated microorganisms, preventing microbial clumping and loss caused by high-speed centrifugation. The microspheres are made of a reversible material, allowing them to dissolve and release the bacteria after solidification, facilitating subsequent experimental procedures. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 This is a flow chart of high-throughput single-cell transcriptome sequencing of microorganisms encapsulated in microspheres in Example 1;

[0048] Figure 2 The microspheres and the microorganisms encapsulated in the microspheres are shown under a bright field microscope after DAPI staining in Example 1;

[0049] Figure 3 The microspheres and the microorganisms encapsulated in the microspheres are shown in the fluorescence field of a microscope after DAPI staining in Example 1;

[0050] Figure 4 are the microspheres in the microsphere suspension in Example 1;

[0051] Figure 5are the microspheres in PBS in Example 1;

[0052] Figure 6 The UMAP (Uniform Manifold Approximation and Projection, a nonlinear dimensionality reduction technique used to map high-dimensional data to a low-dimensional space) map of single-cell sequencing of microsphere-encapsulated intestinal microorganisms obtained in Example 1 using a starting amount of 500,000 microorganisms.

[0053] Figure 7 The existing technology smRandom (CN114507711A) uses a starting amount of 50 million microorganisms to obtain a single-cell sequencing UMAP map of intestinal microorganisms encapsulated in microspheres;

[0054] Figure 8 1 is a comparison chart of the microbial loss rate between Example 1 and the prior art smRandom (CN114507711A);

[0055] Figure 9 This is the quality control image of the library with 10,000 samples loaded in Example 2;

[0056] Figure 10 This is the quality control image of the library with 50,000 samples loaded in Example 2;

[0057] Figure 11 This is the quality control graph of the library with 100,000 samples loaded in Example 2;

[0058] Figure 12 This is the sequencing analysis result of the sample loading amount of 50,000 in Example 2;

[0059] Figure 13 This is the sequencing analysis result of 100,000 samples in Example 2;

[0060] Figure 14 is a microscopic examination graph showing the change in fluorescence brightness over time in different microspheres in Example 3;

[0061] Figure 15 The microspheres encapsulating the microorganisms in Example 4;

[0062] Figure 16 This is a microscope image of the droplet in Example 5. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0064] Example 1: Polyacrylamide hydrogel microspheres encapsulating microbial groups for high-throughput single-cell transcriptome sequencing of microorganisms

[0065] The flowchart of high-throughput single-cell transcriptome sequencing of microorganisms encapsulated in microspheres in this embodiment is as follows: Figure 1 shown.

[0066] Take 1 gram of stool sample and add 5 ml of 4% paraformaldehyde solution by pipetting to mix thoroughly. Use a 40-μm cell sieve to remove larger impurities. Centrifuge at 500g, 4°C for 5 minutes and discard the pellet. Collect the remaining supernatant and place it in a rotary mixer at 10-20 rpm in a refrigerator at 4°C. Fix overnight for 16 hours. After fixation, centrifuge at 4000g, 4°C for 5 minutes to remove the fixative.

[0067] Add 1 ml of PBS containing 1 μl of ribonuclease inhibitor, take 2 μl of bacterial suspension sample, dilute the bacterial suspension sample with 998 μl of water, stain with DAPI (4',6-diamidino-2-phenylindole) fluorescent dye, transfer to a slide, observe and count under a microscope.

[0068] 500,000 microorganisms, 7.5 μl of 40% acrylamide solution, 2.5 μl of 5% N,N'-bis(acryloyl)cystamine, 2.5 μl of 10% ammonium persulfate, and 15 μl of PBS were mixed and then added to 50 μl to prepare a hydrogel microorganism mixture.

[0069] Through a microfluidic chip, 1 ml of oil phase (the oil phase is electronic fluoride liquid 7500 containing 0.2% surfactant) is added with 5 ul of TEMED (N,N,N′,N′-Tetramethylethylenediamine N,N,N′,N′-tetramethylethylenediamine) to shear the hydrogel-microorganism mixture to form oil-in-water droplets of about 25 um in size.

[0070] The droplets were placed at 37°C for 30 minutes to solidify the microspheres. Subsequently, 20% PFO (20% (vol / vol) 1H,1H,2H,2H-Perfluorooctanol in HFE-7500 oil containing 20% volume ratio of 1H,1H,2H,2H-perfluorooctanol dissolved in electronic fluoride liquid NOVEC 7500) was used to break the emulsion and remove the oil phase. The microspheres were washed with n-hexane containing 1% span-80 (Sorbitanoleate (Span 80), sorbitan oleate (Span 80)), and then washed with TBSET buffer (Tris-Buffered Saline-EDTA-Triton Buffer Phosphate Buffer-EDTA-Triton X-100 Buffer). The microspheres were stained with DAPI and examined under a microscope as shown in the figure below. Figure 2 、3 shown.

[0071] The microspheres were placed in PBS buffer containing 0.05% Tween 20 to permeabilize the microbial cell membrane for 5 minutes. The permeabilization reagent was discarded by centrifugation, and lysozyme and lysostaphin were added to digest and lyse the microbial cell wall.

[0072] After lysis, the microspheres were washed two to three times with PBS buffer to remove the lysis reagent. Reverse transcriptase, reaction buffer (containing Tris-HCl, potassium chloride, and magnesium chloride), and random primers with the first round of barcodes were added to capture the total RNA in the bacteria and synthesize cDNA. The random primers were then washed 5-8 times with buffer to remove them.

[0073] The poly(dA) monomers are connected by phosphodiester bonds to form a polydeoxynucleotide chain) tail added in situ to the 3' end of the cDNA using terminal transferase, and then washed with buffer 3-5 times. The microbial microspheres are mixed with density gradient culture medium (iodixanol) to form a microbial microsphere suspension. Compared with PBS, the microbial microspheres in the suspension can be suspended and are not easy to settle, which can avoid clogging of the microfluidic channel. Figure 4 、 5 shown.

[0074] The device was used to encapsulate the microbial microsphere suspension, extension reaction reagent (each 100 μl extension reaction reagent included 7.5 μl DNA polymerase, 7.5 μl ribonuclease H, 7.5 μl USER enzyme (Uracil-Specific Excision Reagen, a chemical reagent used in molecular biology experiments that can specifically recognize and remove uracil (U) residues in DNA molecules), 7.5 μl deoxyribonucleotide triphosphate, 20 μl tenfold concentration polymerase reaction buffer, 50 μl deionized water, and DNA barcode labeled microbeads into droplets of about 100 μm in size.

[0075] The enzymes in the reaction reagents digest the RNA bound to the cDNA in the microorganisms. The DNA barcodes with poly(dT) (polydeoxythymidine) on the microbeads are cleaved and passed through the microbe-encapsulated microbeads before entering the microorganisms. The poly(dT) primer binds to the poly(dA) tail at the end of the cDNA and then extends to add a specific code to the cDNA, adding a unique molecular tag to each cDNA. After these reactions are completed, the oil phase is removed by demulsification with 20% PFO, the microbial microbeads are dissolved with 10mM DTT (dithiothreitol), and the cDNA is purified using DNA purification magnetic beads.

[0076] Finally, PCR (Polymerase Chain Reaction) amplification and sequencing adapters were added to construct a sequencing library. The processed samples were subjected to high-throughput sequencing to analyze the transcriptome information of individual microorganisms. Finally, multiple microbial signals were detected in the fecal samples and clustered. The intestinal microbial data were as follows: Figure 6 As shown, the sequencing volume was 4.1G, and 9762 single bacteria were obtained, with an average of 46 reads (read length) for each bacterium, an average UMI (Unique Molecular Identifiers) number of 33, and an average number of genes of 12.

[0077] The existing technology smRandom-seq (CN114507711A) detected intestinal microbial clustering data in a fecal sample with a starting amount of 50 million microorganisms. Figure 7 As shown in the figure, the sequencing volume was 4.4 GB, and 8026 single bacteria were obtained, with an average of 45 reads per bacterium, an average number of UMIs of 32, and an average number of genes of 13.

[0078] The microbiome samples, Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus were fixed overnight with 4% tissue fixative. The different samples were processed according to the smRandom-seq technical process, and the number of microorganisms remaining after 8 centrifugation cycles (the total number of centrifugations until the completion of the reverse transcription reaction) and 13 centrifugation cycles (the total number of centrifugations until the completion of the poly(dA) tailing reaction) were counted. The loss rate of the reversible hydrogel microspheres encapsulating the microorganisms in Example 1 after the same number of centrifugations was also counted. The microbial loss rates after different treatment methods were compared as shown below. Figure 8 shown.

[0079] from Figure 8 It can be seen that the recovery efficiency of microorganisms after microsphere encapsulation is significantly improved compared to the smRandom-seq technical process. In the smRandom-seq method, the bacterial samples and microbiome samples tested and cultured only retained about 15-60% of the bacteria after centrifugation 8 times (especially the microbiome samples only retained 15% of the bacteria), and after the poly (dA) tailing reaction was completed, the remaining samples only retained 5-30% of the original (the microbiome samples only retained 8% of the bacteria). After the single microorganism was encapsulated in the microspheres by the method of this embodiment and subjected to 13 centrifugation and washing steps, about 90% of the microorganisms could still be recovered, effectively reducing the loss caused by the centrifugation process, confirming that this embodiment can reduce the number of starting microorganisms input.

[0080] The beneficial effects of this embodiment are:

[0081] This example used only 500,000 microorganisms as a test sample. The hydrogel microspheres effectively minimized microbial loss, allowing high-throughput single-cell transcriptome sequencing to be performed even with such a small sample size. Other existing sequencing methods require sample sizes in the tens of millions. Compared to existing methods, the number of microorganisms used in this example is extremely small.

[0082] Example 2: Minimum microbial loading for testing microbiome samples encapsulated in low-melting-point agarose microspheres

[0083] Prepare 1.5% low melting point agarose, fully dissolve it at 85°C for 10 min, and then cool it to 37°C.

[0084] 50 μl of the above agarose solution was added to 100,000 microbial samples treated as in Example 1. Another 50 μl of the agarose solution was added to 50,000 microbial samples treated as in Example 1. Finally, 50 μl of the agarose solution was added to 10,000 microbial samples treated as in Example 1. Agarose solutions with three different microbial concentrations were prepared into approximately 25 μm droplets using the droplet preparation method described in Example 1. The agarose solution was then placed on ice for 15 minutes to solidify.

[0085] Subsequently, the oil phase was removed by demulsification with 20% PFO, and the microspheres were washed with n-hexane containing 1% span80 (Sorbitan oleate (Span80)), and then washed with TBSET buffer.

[0086] The same procedures as in Example 1 were used for subsequent lysozyme treatment, reverse transcription, poly (dA) tailing, in-droplet extension, and coding labeling reactions.

[0087] The quality inspection chart of the library with sample loading of 10,000, 50,000, and 100,000 samples is as follows Figures 9-11 When constructing a normal sample library, the sequence length is mainly distributed between 300-700bp. Figure 9 It can be seen that the main fragments of the sample with a loading of 10,000 are concentrated below 300bp, indicating that most of the sequences in the library construction are amplified by residual primers, and the library construction results are unqualified. Figure 10 、 11 It can be seen that the results of the sample library construction with a sample load of 50,000 microorganisms and 100,000 microorganisms are normal. After being sent to the company for sequencing and analysis of the quality inspection data, more than 1,000 single microorganism information was measured. The samples with a sample load of 50,000 and 100,000 were purified, amplified and constructed into a library. The results are shown in Figure 12 、 13 .

[0088] The beneficial effects of this embodiment are:

[0089] In this embodiment, a sample of 50,000 microorganisms can be used to build a qualified library. Compared with Example 1, this embodiment further reduces the amount of microbial samples. For some small number of precious microbial group samples, the method of the present invention can also be used for high-throughput sequencing.

[0090] Example 3: Permeability test of polyacrylamide hydrogel microspheres with different concentrations

[0091] This example provides polyacrylamide solutions of different concentrations for preparing hydrogel microspheres.

[0092] Concentration 1: 15ul of 40% acrylamide solution, 3.75ul of 5% N,N'-bis(acryloyl)cystamine, 2.5ul of 10% ammonium persulfate, 15ul of PBS, and 10ul of a 150bp DNA fragment (approximately 100kDa) at a concentration of 800ng / ul. Mix and add ultrapure water to 50ul.

[0093] Concentration 2: 15 μl of 40% acrylamide solution, 1.25 μl of 5% N,N'-bis(acryloyl)cystamine, 2.5 μl of 10% ammonium persulfate, 15 μl of PBS, and 10 μl of a 150 bp DNA fragment (approximately 100 kDa) at a concentration of 800 ng / μl. Mix and fill with ultrapure water to 50 μl.

[0094] Concentration 3: 7.5 μl of 40% acrylamide solution, 3.75 μl of 5% N,N'-bis(acryloyl)cystamine, 2.5 μl of 10% ammonium persulfate, 15 μl of PBS, and 10 μl of a 150 bp DNA fragment (approximately 100 kDa) at a concentration of 800 ng / μl. Mix and add ultrapure water to 50 μl.

[0095] Concentration 4: 7.5 μl of 40% acrylamide solution, 2.5 μl of 5% N,N'-bis(acryloyl)cystamine, 2.5 μl of 10% ammonium persulfate, 15 μl of PBS, and 10 μl of a 150 bp DNA fragment (approximately 100 kDa) at a concentration of 800 ng / μl. Mix and fill with ultrapure water to 50 μl.

[0096] Using a microfluidic chip, 1 ml of the oil phase (the oil phase is electronic fluoride solution 7500 containing 0.2% surfactant) is added with 5 μl of TEMED (N,N,N′,N′-Tetramethylethylenediamine N,N,N′,N′-tetramethylethylenediamine) to shear the above-mentioned mixed solutions of different concentrations to form oil-in-water droplets of about 25 μm in size. The droplets are placed in an environment of 37°C for 30 minutes to solidify the microspheres. Four different concentrations of hydrogel microspheres containing substances with a molecular weight of 100 kDa are obtained. Subsequently, 20% concentration of PFO (20% (vol / vol)

[0097] 1H,1H,2H,2H-Perfluorooctanol in HFE-7500 oil (a mixture containing 20% by volume of 1H,1H,2H,2H-perfluorooctanol dissolved in electronic fluorine liquid NOVEC 7500) was demulsified and the oil phase was removed. 1ul of microspheres and 9ul of PBS solution were mixed and immediately stained with DAPI.

[0098] (4',6-diamidino-2-phenylindole 4,6-diamidino-2-phenylindole) fluorescent dye was transferred to a slide and examined under a microscope. The time was started immediately after the microscopic examination, and the changes in the fluorescence brightness of the microspheres of different concentrations were recorded immediately after the microscopic examination, 1min, 3min, 5min, and 7min. The pictures are as follows Figure 14 As shown in the figure, the fluorescent dye is DNA. As the pore size of the microspheres increases, the DNA diffuses out of the microspheres faster. Over time, the amount of fluorescent material inside the microspheres gradually decreases, indicating that the 100 kDa substance can freely enter and exit the microspheres. At concentration 4, the amount of fluorescent material inside the microspheres has significantly decreased after 1 minute, and is completely gone after 3 minutes, indicating that the microspheres produced at concentration 4 have very good permeability.

[0099] The beneficial effects of this embodiment are:

[0100] The hydrogel microspheres made of polyacrylamide have good permeability, and substances of 100kDa can freely enter and exit the microspheres. The proteins and reagents required for the single-cell sequencing process can also freely enter and exit.

[0101] Example 4: Labeling cDNA after dissolution of microspheres

[0102] In this example, 150,000 microbial samples, 7.5 μl of 40% acrylamide solution, 2.5 μl of 5% N,N'-bis(acryloyl)cystamine, 2.5 μl of 10% ammonium persulfate, and 15 μl of PBS were mixed and then diluted to 50 μl to prepare a hydrogel microbial mixture.

[0103] Add 2.5ul of TEMED (N,N,N′,N′-Tetramethylethylenediamine) to 50ul of the oil phase (the oil phase is electronic fluoride solution 7500 containing 0.2% surfactant) in a 1.5ml centrifuge tube containing 50ul of the hydrogel-microorganism mixture, and place the centrifuge tube on a vortex shaker for 2 minutes to form oil-in-water droplets of varying sizes, such as Figure 15 shown.

[0104] Demulsification, cell wall lysis, reverse transcription, and poly(dA) addition were performed using the same methods as in Example 1. After the poly(dA) tailing reaction was completed, the microspheres were washed 3-5 times with buffer and then dissolved in DTT at a final concentration of 10 mM. The microorganisms were mixed with a density gradient medium (iodixanol) to form a microbial suspension. Subsequent processing was performed as in Example 1, with the final demulsification using 20% PFO to remove the oil phase, and cDNA was purified using DNA purification magnetic beads.

[0105] The beneficial effects of this embodiment are:

[0106] In this embodiment, the microspheres are dissolved first and then labeled, and the labeling action is not limited to occur inside the microspheres.

[0107] Example 5: Simultaneous dissolution of microspheres and RNA labeling

[0108] The operation method of this embodiment is the same as that of embodiment 1 except for the following steps.

[0109] The device was used to encapsulate the microbial microsphere suspension, extension reaction reagent (each 100 μl extension reaction reagent included 10 ul 100 mM DTT, 7.5 μl DNA polymerase, 7.5 μl RNase H, 7.5 μl USER enzyme (Uracil-Specific Excision Reagent, a chemical reagent used in molecular biology experiments that can specifically recognize and remove uracil (U) residues in DNA molecules), 7.5 μl deoxyribonucleotide triphosphate, 20 μl ten-fold concentration polymerase reaction buffer, 50 μl deionized water, and DNA barcode labeled microbeads into a droplet of about 100 μm in size, such as Figure 16 As shown in Figure 2, in the microfluidic chip, the reagents and hydrogel microspheres enter through separate channels, preventing the hydrogel microspheres from being dissolved by DTT. After the oil phase is sheared to form droplets, the hydrogel microspheres and the extension reaction reagents containing DTT are encapsulated in the droplets and dissolved.

[0110] After the reaction was completed, 20% PFO was used to break the emulsion and remove the oil phase, and cDNA was purified using DNA purification magnetic beads.

[0111] The beneficial effects of this embodiment are:

[0112] In this embodiment, the reagents, DNA-encoded microspheres, and hydrogel microspheres are encapsulated together in the droplets, and the dissolution and encoding of the hydrogel microspheres are carried out simultaneously in the droplets.

[0113] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A microbial single-cell transcriptome sequencing method based on hydrogel microspheres, characterized in that: include: The microbial sample to be tested is fixed and then encapsulated in hydrogel microspheres; cDNA is synthesized from the RNA of a single microbial cell inside the microsphere and the cDNA is labeled; a library is constructed and sequenced to obtain transcriptome information of the single microbial cell; The sequencing method specifically comprises the following steps: preparing the microbial sample into a single cell suspension, and uniformly mixing the suspension with a fixative to obtain a fixed microbial sample; mixing the fixed microbial sample and the hydrogel solution to obtain a hydrogel solution containing the microbial sample; preparing the hydrogel solution containing the microbial sample into water-in-oil droplets, and solidifying the hydrogel solution in the droplets; Demulsifying the water-in-oil droplets and washing to obtain the microspheres encapsulating the microbial sample; digesting and lysing the cell walls of the microbial sample in the microspheres so that the reagent can pass through the cell walls and enter the cells of the microbial sample; Performing an in situ reverse transcription reaction on the RNA of the microbial cell using a reverse transcription primer to synthesize the first strand of cDNA, and then labeling the cDNA; dissolving the microspheres, purifying the cDNA released from the microspheres, and constructing a library and sequencing the purified cDNA to obtain transcriptome information of the microbial single cell; Wherein, there is no or only one microorganism in the microsphere, and the microsphere has a porous network structure, and the holes on the surface can pass the reagent and block the microorganisms in the microsphere; The preparation method of the hydrogel solution is as follows: Mix 7.5 μL of 40% acrylamide solution, 2.5 μL of 5% N,N'-bis(acryloyl)cystamine solution, 2.5 μL of 10% ammonium persulfate solution, and 15 μL of PBS, and then add ultrapure water to 50 μL. Alternatively, mix 15 μL of 40% acrylamide solution, 1.25 μL of 5% N,N'-bis(acryloyl)cystamine solution, 2.5 μL of 10% ammonium persulfate solution, and 15 μL of PBS, and then add ultrapure water to 50 μL. Alternatively, mix 7.5 μL of 40% acrylamide solution, 3.75 μL of 5% N,N'-bis(acryloyl)cystamine solution, 2.5 μL of 10% ammonium persulfate solution, and 15 μL of PBS, and then add ultrapure water to 50 μL. Alternatively, fully dissolve 1.5% low-melting-point agarose at 85°C for 10 minutes and then cool to 37°C.

2. The microbial single-cell transcriptome sequencing method based on hydrogel microspheres according to claim 1, characterized in that: The fixative is a simple fixative or a mixed fixative; The simple fixative comprises one of paraformaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate and mercuric chloride; The mixed fixative includes multiple types of acetic acid-alcohol mixed solution, formalin-acetic acid-alcohol solution and Boyne's fixative.

3. The microbial single-cell transcriptome sequencing method based on hydrogel microspheres according to claim 1, characterized in that: The hydrogel microspheres can be dissolved after solidification.

4. The microbial single-cell transcriptome sequencing method based on hydrogel microspheres according to claim 1, characterized in that: The method for preparing the water-in-oil droplets comprises: Microfluidics, batch emulsification, 3D printing, photolithography, and electrospraying.

5. The microbial single-cell transcriptome sequencing method based on hydrogel microspheres according to claim 1, characterized in that: The method for labeling the cDNA comprises: A barcoded reverse transcription primer is used to capture all RNA in the microbial sample, and the first chain of cDNA is synthesized in situ by reverse transcription on the RNA, and a first round of labeling is added at the same time, followed by one or more labels.

6. The microbial single-cell transcriptome sequencing method based on hydrogel microspheres according to claim 1, characterized in that: The method for labeling the cDNA comprises: RNA is polyadenylated in situ using Poly (A) polymerase I, and random hexamer primers with barcode labels and poly (dT) primers with barcode labels are used to perform the first round of labeling and reverse transcription reaction on microorganisms to synthesize the first chain of cDNA. The second and third rounds of barcode labeling are then performed using ligase, and finally the cDNA is purified and the second chain of cDNA is synthesized.

Images