Method for single-cell transcriptome sequencing

Abstract

Disclosed in the present invention is a method for single-cell transcriptome sequencing. The method comprises: mixing microorganisms with an encapsulation material, solidifying same to form a jelly-like semi-solid, and digesting the cell walls with lysozyme to permeabilize the bacteria; adding a reverse transcription reagent and a random primer to capture total RNA in the bacteria and synthesize cDNA; dissolving the encapsulation material to release individual bacterial cells, and encapsulating the individual bacterial cells and labeled microbeads into droplets using a microfluidic device; enzymatically digesting RNA in the bacteria to release the cDNA from the bacteria, collecting and purifying the cDNA after demulsification, performing amplification, and adding sequencing adapters to construct a sequencing library; and digesting cDNA products derived from rRNA, and enriching cDNA products derived from mRNA. High-throughput sequencing is performed on the processed samples to analyze transcriptome information of individual microorganisms. According to the present invention, the microorganisms are encapsulated using a reversibly solidifiable porous material without centrifugal washing. The present invention facilitates single-cell analysis of microbial samples with a small initial amount.

Description

Single bacterial transcriptome sequencing method TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to transcriptome sequencing, more specifically to a single bacterial transcriptome sequencing method. BACKGROUND

[0002] Prior art smRandom-seq (patent application number: CN202210174619.9) first fixes the separated microbial sample, 4% paraformaldehyde is used to fix the bacteria overnight to cross-link the RNA, DNA and protein inside the bacteria. The cell wall is digested using lysozyme, and the fixed bacteria are permeabilized to facilitate the next in situ reverse transcription reaction. The microorganism is used as a reaction container for in situ reaction, random primers are added to bind with the RNA inside the bacteria, total RNA is captured to synthesize cDNA by reverse transcription, and a poly(dA) tail is added to the 3' end of the cDNA in situ by terminal transferase (TdT). After each step of the foregoing process is completed, the buffer needs to be washed for about 3-5 times to prevent the influence of reagent residues on the subsequent reaction. A microfluidic device is used to encapsulate a single bacterium and labeled microbeads into a droplet. The poly(T) primer is released from the microbeads by enzyme digestion, the RNA in the bacteria is digested to release the cDNA from the bacteria, the poly(T) primer is combined with the poly(A) tail at the end of the cDNA, and then the cDNA is added with a specific code and a molecular tag (UMI). After demulsification, the purified cDNA is collected, amplified and added with sequencing adapters to construct a sequencing library, the cDNA product of rRNA is digested by cas9, and the cDNA product of mRNA is enriched. High-throughput sequencing is performed on the treated sample to analyze the transcriptome message of a single microorganism.

[0003] Existing microbial single-cell sequencing technologies: smRandom-seq (Patent application number: CN202210174619.9); MicroSPLiT (microbial split-pool ligation transcriptomics, Kuchina A, Brettner LM, Paleologu L, Roco CM, Rosenberg AB, Carignano A, et al. M icrobial single-cell RNA sequencing by split-pool barcoding. Science 2021; 371(6531): eaba5257.); PETRI-seq (prokaryotic expression profiling by tagging RNA in situ and sequencing, Blattman SB, Jiang W, Oikonomou P, Tavazoie S. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. Nat Microbiol 2020; 5(10): 1192-201.).

[0004] Microorganisms are relatively small, and a high centrifugal speed (~4000g) is required for centrifugation. Multiple centrifugation and washing can easily lead to the loss of microorganisms, and high-speed centrifugation can cause bacterial adhesion, which can cause double bacterial contamination. The existing technology requires a high starting amount for single-cell analysis of microorganisms. The amount of microorganisms collected in clinical practice is often small, and culture amplification can affect the composition and state of microorganisms, so single-cell analysis of microorganisms is often not possible.

[0005] PETRI-seq and MicroSPLIT are combination-indexing based techniques, which perform reverse transcription (RT) after bacterial immobilization and permeabilization. In PETRI-seq, random primers are used for reverse transcription; while in MicroSPLIT, transcripts are polyadenylated (poly(A)-tailed) before reverse transcription. RT is performed with ploy(T) primers. Both methods perform multiple rounds of indexing tag ligation after RT (bacteria are first distributed to different reaction wells to ligate well-specific tags, then collected, mixed and distributed to different reaction wells to ligate well-specific tags, and repeated multiple times), which ligate specific sequential barcode labels (barcode sequence is the position sequence of a certain bacterium in multiple distribution) on cDNA, giving each individual bacterium a unique identity. The process also has a large number of centrifugal washing processes, including RT and each round of tag ligation (2-3 rounds). The sample starting amount has a large requirement (~100 million microorganisms), while the present application has a small starting amount (~100,000 microorganisms), which can meet more microbial single bacterium analysis scenarios. SUMMARY

[0006] In view of the defects of the prior art, the microorganism sample is first fixed, and the fixed bacterial sample is wrapped using reversible porous material (having reversible characteristics, which can be in solid or liquid state under different conditions; porous, the material has internal pores, allowing biological macromolecules to pass through). The wrapping material can be at least one of natural biological macromolecules (such as agarose, gelatin, sodium alginate, hyaluronic acid) or artificially synthesized materials (such as at least one of polyethylene glycol and polyacrylamide). The microorganism and the wrapping material are mixed and placed in a mold to form a jelly-like semi-solid after solidification. The wrapping material has pores in the middle, which can allow macromolecular enzymes, salt ions, and water to pass through, but can lock the single microorganism in the wrapping material. After wrapping, the wrapped body is transferred to a centrifuge tube or a special chip for cleaning and reaction. Since the microorganism has been locked in the wrapped body, the centrifuge does not need to be used for cleaning. The cell wall is digested using lysozyme to permeabilize the bacteria. Reverse transcription reagents and random primers are added to capture and synthesize cDNA from total RNA in the bacteria. A poly(dA) tail is added to the 3' end of the cDNA in situ by terminal transferase (TdT). After each step of the foregoing process is completed, the buffer is used for cleaning about 3 times to prevent the influence of residual reagents on the subsequent reaction. The wrapping material is dissolved to release the single bacteria, which are encapsulated in droplets with labeled microbeads using a microfluidic device after cleaning. The poly(T) primer is released from the microbead by enzyme digestion, and the RNA in the bacteria is digested to release the cDNA from the bacteria. The poly(T) primer is combined with the poly(A) tail at the end of the cDNA, and then the cDNA is added with a specific code and a molecular tag (UMI). After demulsification, the purified cDNA is collected, amplified, and added with a sequencing adapter to construct a sequencing library. The cDNA product of rRNA is digested by cas9, and the cDNA product of mRNA is enriched. The treated sample is subjected to high-throughput sequencing to analyze the transcriptome information of a single microorganism.

[0007] The present application provides a single-bacterium transcriptome sequencing method, which comprises the following steps:

[0008] The microorganism is fixed and wrapped using reversible solidification porous material to form a wrapped body, and the capture and adapter addition of bacterial RNA are completed in the wrapped body. The single bacterium is released, and the treated sample is subjected to high-throughput sequencing to analyze the transcriptome information of a single microorganism.

[0009] The wrapping material has pores in the middle, which can allow macromolecular enzymes, salt ions, and water to pass through, but the single microorganism is locked in the wrapping material.

[0010] Specifically, the method comprises the following steps:

[0011] S1 fixes the microbial sample, and uses a reversible porous material to wrap the fixed microbial sample, and the wrapping material is selected from natural biological macromolecules or artificial synthetic materials;

[0012] S2, after the wrapping is completed, the wrapped body is cleaned, the cell wall is digested using lysozyme, and the microorganism is permeabilized;

[0013] S3, the RNA in the microorganism is labeled, specifically, the wrapped body sample is soaked in a labeling reagent, the labeling reagent includes a nucleotide chain, a tool enzyme and a reaction buffer, the nucleotide chain is combined with the RNA fragment, and after the nucleotide is combined with the RNA chain, a polymerization reaction or a connection reaction occurs, so that the nucleotide chain carries the RNA information and a capture linker; after completion, cleaning is performed;

[0014] S4, the wrapping material is dissolved to release single bacteria, and after cleaning, single bacteria are separated using a microfluidic droplet or a microwell plate system to obtain single droplets;

[0015] S5, the single droplets after the single bacteria separation are subjected to an extension reaction, the single droplets are broken, cDNA is purified, and PCR amplification is performed;

[0016] S6, a sequencing library is constructed, and sequencing is performed to analyze the transcriptome information of a single microorganism.

[0017] Preferably, the reversible porous material has reversible characteristics and can be in a solid state or a liquid under different conditions; and the reversible porous material has internal pores allowing biological macromolecules to pass through. Specifically, the natural biological macromolecule is selected from at least one of agarose, gelatin, sodium alginate and hyaluronic acid; and the artificial synthetic material is selected from at least one of polyethylene glycol or polyacrylamide.

[0018] Further preferably, in S3, the RNA of the microbial sample is labeled by a reverse transcriptase, specifically, the RNA is labeled by adding a reverse transcriptase, a nucleotide chain and a reaction buffer, and a capture linker is added by adding an end transferase and a deoxyribonucleoside triphosphate.

[0019] In a specific embodiment, in S4, the single bacteria separation step is performed using a microfluidic droplet system as follows: the microbial sample, the extension reaction reagent, the coded microsphere and the oil phase are respectively connected with the corresponding liquid inlet of the microfluidic chip to form a water-in-oil single droplet containing single bacteria, single coded microspheres and extension reaction reagents, the single droplet is collected, and a single chamber containing single bacteria is formed;

[0020] In a specific embodiment, in S5, the collected single droplets are subjected to an extension reaction to synthesize a second strand of cDNA with a barcode label in the single droplet; after the extension reaction is completed, the single droplets are broken, the cDNA in the extraction tube is purified, and PCR amplification is performed.

[0021] In step S6, the cDNA amplified in step S5 is subjected to end repair and A tailing by using a TA cloning ligation adapter library method, and the ligation adapter is connected to the library by using a library reagent to construct a sequencing library. The cDNA product of the rRNA is digested by using cas9, and the cDNA product of the mRNA is enriched. The constructed library is subjected to high-throughput sequencing by using an Illumina sequencing platform to analyze the transcriptome information of a single microorganism.

[0022] Preferably, the washing does not require centrifugation, and preferably, the washing is washing with a buffer to prevent the influence of reagent residues on subsequent reactions. The specific operation is washing 2-4 times with PBST.

[0023] Optionally, a data analysis step is further included, and specifically, the data of the detected microorganism is clustered.

[0024] Specifically, the microorganism sample is an environmental microorganism sample or a microorganism sample in excrement or body fluid of an organism.

[0025] More specifically, the microorganism is a bacterium. In a specific embodiment, the amount of the microorganism is not less than 4000 bacterial cells or preferably not less than 5000 bacterial cells per 50 microliters.

[0026] The advantages of the method are as follows: the microorganism is wrapped with a reversible porous material to form a wrapped body, centrifugal washing is changed into non-centrifugal washing, the microorganism completes the washing and RNA capture labeling reaction in the wrapped body, the recovery efficiency of the microorganism is improved, the adhesion ratio of the microorganism is reduced, the starting input amount of the microorganism is reduced, and single bacterial analysis of a small amount (~100,000) of a microorganism sample is realized. And the non-centrifugal washing is more difficult to realize automatically. The microorganism is wrapped with a reversible coagulation porous material before the microorganism is operated, and the capture and adapter addition of bacterial RNA are completed in the wrapped body. Thus, the centrifugal washing between reactions is changed into non-centrifugal washing, the microorganism agglomeration caused by high-speed centrifugation is prevented, the loss of the microorganism caused by centrifugation is reduced, and the proportion change of the bacteria caused by centrifugation is reduced. The present application can perform single bacterial analysis on a small amount of a microorganism sample. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a wrapped body mold diagram.

[0028] Figure 2 is a wrapped body schematic diagram.

[0029] Figure 3 is a wrapped body microorganism staining result diagram.

[0030] Figure 4 is an agarose-wrapped single bacterial microorganism sequencing result.

[0031] Figure 5 Sequencing results of single bacterial microorganism wrapped with polyacrylamide.

[0032] Figure 6 Comparison of single bacterial recovery efficiency.

[0033] Figure 7 Sequencing results of single bacterial microorganism with low starting amount. DETAILED DESCRIPTION

[0034] The present application is described below by way of specific examples for better understanding of the present application, but does not constitute a limitation on the present application.

[0035] Example 1: Low-melting agarose wrapped intestinal microorganism sample for high-throughput single bacterial transcriptome sequencing

[0036] Step one: Microorganism fixation: Take about 1 gram of fecal sample, add 5 milliliters of 4% PFA (polyformaldehyde), and mix well by blowing. Put the mixture into a rotating mixer in a 4°C refrigerator, rotate at 10-20 rpm overnight for 12 hours. After fixation, remove the larger impurities by passing the liquid through a 40-micron cell screen, collect in a 50-milliliter centrifuge tube, centrifuge at 500g, 4°C, for 2 minutes, collect the supernatant, pass it through a 10-micron cell screen, transfer to a 15-milliliter centrifuge tube, centrifuge at 3900g, 4°C, for 5 minutes to remove the fixation liquid. Add 1 milliliter of PBS containing 1 microliter of RNase inhibitor, take 2 microliters of bacterial suspension sample, dilute with 998 microliters of water, PI fluorescent dye staining, transfer to a glass slide, and observe under a microscope at 200x field of view for subsequent experimental cell loss and morphological change comparison and evaluation.

[0037] Step two: Microorganism wrapping: Take 1 million bacteria, centrifuge at 3900g, 4°C, for 5 minutes to remove the supernatant. Melt 1.5% low-melting agarose at 85°C and let it cool to room temperature. Take 50 microliters of agarose to suspend the bacteria, and transfer to a solidification mold (Figure 1), and let it stand on ice for 15 minutes to solidify.

[0038] Step three: Microorganism permeabilization: Take the package out of the mold, the package is semi-solid, can be transferred to a 200 microliter centrifuge tube with tweezers (Figure 2), the microorganism is randomly distributed and inlaid in the package (Figure 3), soak in PBS containing RNase inhibitor, and wait on ice. Prepare cell wall digestion reagent (high-throughput intestinal flora single bacterial transcriptome kit from Hangzhou Yuezhen Biotechnology Co., Ltd.). Aspirate the PBS in the centrifuge tube, add 200 microliters of pre-cooled PBST, and stand on ice for 5 minutes for permeabilization. Add 800 microliters of PBS, centrifuge, aspirate the supernatant, and add 200 microliters of cell wall digestion solution. Place the centrifuge tube in a 37°C, 800 rpm metal bath, react for 15 minutes, immediately take out and place on ice, aspirate the supernatant, add 200 microliters of pre-cooled PBST to terminate digestion, soak for 30 seconds, remove the supernatant, add 200 microliters of pre-cooled PBST for washing twice, and add 200 microliters of PBST on ice for standby.

[0039] Step four: RNA labeling: Prepare reverse transcription reagent (high-throughput intestinal flora single bacterial transcriptome kit from Hangzhou Yuezhen Biotechnology Co., Ltd.), 50 microliters of reverse transcription reagent (17.50 microliters of Buffer RN, 12.60 microliters of Buffer B2, 3.5 microliters of Buffer TX, 3.5 microliters of Enzyme R, 3.5 microliters of RNase inhibitor, and 10 microliters of random reverse primer). Aspirate the supernatant in the centrifuge tube, add the reverse transcription reagent, and place the centrifuge tube in a PCR instrument for gradient temperature cycling (8°C to 42°C, cycle 11 times) reverse transcription reaction. After the reaction is completed, aspirate the supernatant, add 200 microliters of washing solution for washing three times, and add 200 microliters of washing solution on ice for standby. Prepare tailing reagent (high-throughput intestinal flora single bacterial transcriptome kit from Hangzhou Yuezhen Biotechnology Co., Ltd.), 50 microliters of A-tailing reagent (5 microliters of Buffer T1, 5 microliters of Buffer T2, 0.5 microliters of Adapter, 0.5 microliters of Enzyme T, and 39 microliters of Buffer W). Aspirate the supernatant in the centrifuge tube, add the A-tailing reagent, and place the centrifuge tube in a PCR instrument for 37°C incubation for 30 minutes. After the reaction is completed, aspirate the supernatant, and add 200 microliters of washing solution for soaking and washing three times.

[0040] Step five: Package dissolution and release of microorganisms: After washing, transfer the package to a 1.5 milliliter centrifuge tube, place in a 65°C metal bath for 5 minutes, add 1 ml of washing solution, and mix well by blowing. Centrifuge at 3900g, 4°C, for 5 minutes to remove the supernatant. Mix 2 microliters of bacterial suspension with 38 microliters of water, and then observe under a microscope, count, and observe the state of the bacteria at this time.

[0041] Step six microfluidic package: configure microfluidic reagent (high-throughput intestinal flora single bacterial transcriptome kit of Hangzhou Yuezhen Biotechnology Co., Ltd.) to mix 100,000 bacteria (60 microliters), 30 microliters of OptiPrep (sigma) and 30 microliters of extension reaction reagent into the suspension for loading. Prepare the microfluidic chip, add 120 microliters of the suspension for loading to the suspension for loading bin, add 55 microliters of the labeled microspheres to the microsphere bin, and add oil to the oil phase stock solution bin. After the sample loading is completed, cover the chip with a sealing silica gel pad, which should completely cover the upper surface of the chip, and the surface after covering should be flat without tilting. Put the chip into the VITAcruizer single cell preparation instrument (Hangzhou Yuezhen Biotechnology Co., Ltd.) to generate microdroplets. Collect the droplets into a 200 μl centrifuge tube and place it in a PCR instrument for reaction (program: 37°C for 1 hour, 50°C for 30 minutes, 60°C for 30 minutes, 75°C for 20 minutes) to perform extension reaction.

[0042] Step seven sequencing library construction: after the extension reaction is completed, PFO (perfluorooctanesulfonate) is added to the microdroplets, which releases the cDNA in the microemulsion droplets by destroying the surfactant molecular film of the oil phase shell. The cDNA is purified and extracted by magnetic bead method. Part of the double-stranded cDNA is taken as a template for qPCR experiment, and the total cDNA content captured is evaluated by CT value. The remaining cDNA is subjected to PCR amplification, and the amplified cDNA is subjected to end repair and A tailing using TA cloning adapter ligation library construction method. The constructed library is added with cas9 probe and enzyme to digest the rRNA cDNA product, and high-throughput sequencing is performed using Illumina sequencing platform.

[0043] Step eight data analysis: the detected intestinal microbial data is shown in Figure 4, and a variety of microbial signals can be detected and clustered in the fecal sample. The sequencing amount is 13.7G, 4775 single bacteria are obtained, the average number of reads per bacterium is 562, the average UMI number is 257, and the average gene number is 38.

[0044] Example 2: use of polyacrylamide to package microorganisms

[0045] According to step one of example 1, the microorganisms are fixed.

[0046] Step two Microbe encapsulation: Prepare acrylamide material and buffer according to the paper Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding (Advanced Science 2020). Add 50 microliters of TBSET buffer, 30 microliters of 10% (w / v) APS (Sigma-Aldrich, A9164), 75 microliters of 40% (v / v) Acrylamide solution (Sigma-Aldrich, A4058-100ML), 245 microliters of 0.8% (w / v) BAC (Sigma-Aldrich, A4929-5G) and 100 microliters of double distilled water in a centrifuge tube, mix well, take 50 microliters, suspend 1 million bacteria, add 0.5ul TEMED, mix well. Place in mold, 40°C for 30 min to solidify.

[0047] According to Example 1 Step three, perform microbial permeabilization, and Step four, perform RNA labeling.

[0048] Step five Encapsulated body dissolution to release microbes: After the reaction, the encapsulated body is washed and placed in a centrifuge tube, 500 microliters of 1 millimolar DTT is added to dissolve the encapsulated body. Add 1 milliliter of washing solution and mix well by blowing. Remove the supernatant by centrifugation at 3900g, 4°C for 5 minutes. Take 2ul of bacterial suspension and mix with 38ul of water, then observe under a microscope, count and observe the state of the bacteria at this time.

[0049] According to Example 1 Step six, perform microfluidic encapsulation, and Step seven, perform sequencing library construction.

[0050] Step eight Data analysis: The detected intestinal microbial data is shown in Figure 5, and a variety of microbial signals can be detected and clustered in the fecal sample. The sequencing amount is 9.5G, 3098 single bacteria are obtained, the average number of reads per bacterium is 386, the average UMI number is 180, and the average gene number is 21.

[0051] Example 3: Comparison of bacterial recovery rate of the present application and smRandom-seq

[0052] Microbial samples were prepared and counted using the smRandom-seq method published in Nature communications 2023 (Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq). Compared with the recovery efficiency of the protocol microbial samples in Examples 1 and 2, the recovery efficiency of the wrapped microbial samples was significantly improved (Figure 6). In the smRandom-seq method, a large amount of centrifugal washing was performed on the microbial samples, and about 60% of the bacteria remained after RT. After A-tailing, only about 40% of the bacteria remained. Through the method of the present application, about 90% of the bacteria can be recovered after wrapping, washing, and A-tailing, reducing the number of starting microorganisms for analysis.

[0053] Example 4: Single-microbial transcriptome sequencing with low starting amount

[0054] The fecal sample was used to fix the microorganisms according to Step One of Example 1.

[0055] Step Two: Wrap the microorganisms according to the previous description: In a 50-microliter volume, take gradient dilution of microorganisms to 100,000, 50,000, 25,000, and 5,000 bacterial cells, respectively. Melt 1.5% low-melting-point agarose at 85°C and let it cool to room temperature. Take 50 microliters of agarose suspension of bacteria and transfer it to a coagulation tool (Figure 1). Let it coagulate on ice for 15 minutes.

[0056] According to Steps Three, Four, Five, Six, and Seven of Example 1, perform microbial permeabilization, RNA labeling, wrapped body dissolution to release microorganisms, microbial microfluidic wrapping, and sequencing library construction.

[0057] Step Eight: Data Analysis: The sample sequencing data is shown in Table 1, and the detected intestinal microbial data is shown in Figure 7.

[0058] Table 1

[0059] Based on Table 1 and Figure 7, the recovery effect of the protocol of the present application is stable, and when the starting amount of microorganisms is reduced to 5,000 bacteria, a variety of microbial signals can still be detected and clustered in the fecal sample. The starting amount of microorganisms is reduced from 100,000 to 5,000 bacteria, and the detected microbial species are consistent, indicating that the sample bias produced during the recovery process is small. In addition, the 0.5-watt sample sequencing is close to saturation, the starting amount and the number of microorganisms obtained are close to 10:1, which is consistent with the 10% sample wrapping rate produced by the microfluidic wrapping process in Step Six.

Claims

1. A method of single bacterial transcriptome sequencing, characterized in that, It comprises the following steps: After the microorganism is fixed, it is wrapped with reversible coagulation porous material to form a wrapped body, and the capture of the microorganism RNA and the addition of the linker are completed in the wrapped body, and then the single bacteria are released, and the treated sample is subjected to high-throughput sequencing to analyze the transcriptome information of the single microorganism. The wrapped material has pores in the middle, which can allow macromolecular enzymes, salt ions, and water to pass through, but the single microorganism is locked in the wrapped material.

2. The single bacterial transcriptome sequencing method of claim 1, wherein, It comprises the following steps: S1: Fix the microorganism sample, and wrap the fixed microorganism sample with reversible porous material, and the wrapping material is selected from natural biological macromolecules or artificial synthetic materials; S2: After the wrapping is completed, the wrapped body is washed, the cell wall is digested with lysozyme, and the microorganism is permeabilized; S3: Label the RNA in the microorganism, specifically, immerse the wrapped body sample in a labeling reagent, the labeling reagent includes a nucleotide chain, a tool enzyme, and a reaction buffer, combine the nucleotide chain with the RNA fragment, and after the nucleotide is combined with the RNA chain, a polymerization reaction or a connection reaction occurs, so that the nucleotide chain carries the RNA information and the capture linker; after completion, wash; S4: Dissolve the wrapped material to release the single bacteria, and after washing, use a microfluidic droplet or a microwell plate system to divide the single bacteria to obtain single droplets; S5: Perform extension reaction on the single droplets after single bacteria division, break the single droplets, purify cDNA, and perform PCR amplification; S6: Construct a sequencing library and perform sequencing to analyze the transcriptome information of the single microorganism.

3. The single bacterial transcriptome sequencing method of claim 2, wherein, The reversible porous material has reversible characteristics and can be in a solid or liquid state under different conditions, and the internal pores of the reversible porous material allow biological macromolecules to pass through.

4. The single bacterial transcriptome sequencing method of claim 2, wherein, The natural biological macromolecule is selected from at least one of agarose, gelatin, sodium alginate, and hyaluronic acid; and the artificial synthetic material is selected from at least one of polyethylene glycol or polyacrylamide.

5. The single bacterial transcriptome sequencing method of claim 2, wherein, In S3, the RNA of the microorganism sample is labeled by reverse transcriptase, specifically by adding reverse transcriptase, a nucleotide chain, and a reaction buffer for RNA labeling, and by adding terminal transferase and a deoxyribonucleoside triphosphate for capture linker addition.

6. The single bacterial transcriptome sequencing method of claim 2, wherein, In S4, the single bacteria division step using a microfluidic droplet system is as follows: connect the microorganism sample, the extension reaction reagent, the coded microsphere, and the oil phase to the corresponding liquid inlet of the microfluidic chip respectively to form a water-in-oil single droplet containing single bacteria, single coded microspheres, and extension reaction reagents, collect the single droplets, and form a single chamber containing single bacteria.

7. The single bacterial transcriptome sequencing method of claim 2, wherein, In S5, the collected single droplets are subjected to extension reaction to synthesize a second strand of cDNA with a barcode label in the single droplet; after the extension reaction is completed, the single droplets are broken, the cDNA in the extraction tube is purified, and PCR amplification is performed; In step S6, the cDNA amplified in step S5 is subjected to end repair and A tailing by using a TA cloning adaptor ligation library method, and the adaptor is connected to the library by using a library construction kit to construct a sequencing library, the cDNA product is digested by cas9 to eliminate rRNA, and the cDNA product is enriched to mRNA; the constructed library is subjected to high-throughput sequencing by using an Illumina sequencing platform to analyze the transcriptome information of a single microorganism.

8. The single bacterial transcriptome sequencing method of claim 2, wherein, The washing does not need centrifugation, and preferably, the washing is washing with a buffer solution to prevent the influence of reagent residues on subsequent reactions; and the specific operation is washing 2-4 times with PBST.

9. The single bacterial transcriptome sequencing method of claim 7, wherein, The method further comprises a data analysis step, specifically, clustering of the detected microorganism data.

10. The single bacterial transcriptome sequencing method of any one of claims 1 to 9, wherein, The microorganism sample is an environmental microorganism sample or a microorganism sample in biological excrement or body fluid.

11. The single bacterial transcriptome sequencing method of claim 10, wherein, The microorganism is bacteria.

12. The single bacterial transcriptome sequencing method of claim 10, wherein, The amount of the microorganism is not less than 4000 bacterial cells, preferably not less than 5000 bacterial cells, per 50 microliters.

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