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CN-121975911-A - Pretreatment method of microbial single-cell sequencing sample and microbial single-cell RNA sequencing method

CN121975911ACN 121975911 ACN121975911 ACN 121975911ACN-121975911-A

Abstract

The invention discloses a pretreatment method of a microbial single-cell sequencing sample and a microbial single-cell RNA sequencing method, wherein the pretreatment method is carried out in a sampling tube, a sample fixing cavity and at least one independent cavity isolated from the sample fixing cavity are arranged in the sampling tube, a fixing liquid is pre-arranged in the sample fixing cavity or the independent cavity, a diluent is pre-arranged in the independent cavity, and at least one of the fixing liquid and the diluent contains a nucleic acid protecting agent. The method comprises the steps of sampling a sample, sealing a sampling tube, mixing the sample with a fixing liquid under a sealing condition for fixing, and mixing a diluent and the fixing liquid in the sampling tube before, simultaneously or after fixing, so that the diluted fixing liquid and the sample coexist in the sampling tube for fixing and preserving, or diluting the fixing liquid in the sampling tube after fixing is finished for preserving. The fixing and preserving processes are all completed in the closed sampling tube.

Inventors

  • WANG YONGCHENG
  • DING LIGUO

Assignees

  • 浙江大学

Dates

Publication Date
20260505
Application Date
20251219

Claims (13)

  1. 1. The pretreatment method of the microbial single-cell sequencing sample is characterized in that the pretreatment method is carried out in a sampling tube, a sample fixing cavity for containing the sample and fixing the sample and at least one independent cavity isolated from the sample fixing cavity are arranged in the sampling tube, diluent is pre-contained in the at least one independent cavity, fixing liquid is pre-contained in the sample fixing cavity or the at least one independent cavity, and at least one of the fixing liquid and the diluent contains a nucleic acid protecting agent; the pretreatment method comprises the following steps: S1, sampling a sample into a sampling tube and closing the sampling tube; s2, mixing the sample with a fixing solution in the closed sampling tube simultaneously or subsequently to the step S1 to fix the cells; and S3, mixing the diluent with the fixing solution to dilute the fixing solution before, simultaneously with or after the step S2 in the closed sampling tube.
  2. 2. The pretreatment method according to claim 1, wherein the sampling tube comprises a tube body (1) and a cover body (2), and the cover body (2) is provided with a connecting part (21) detachably connected with the tube body (1); The sample fixing cavity is arranged in the pipe body (1), the independent cavity comprises a first independent cavity arranged in the cover body (2), the first independent cavity is communicated with the sample fixing cavity through a flow channel (23), the flow channel (23) is connected with the connecting part (21) and is internally provided with a diaphragm (26), and the diaphragm (26) is provided with a closing position for closing the flow channel (23) and an opening position for opening the flow channel (23) due to pressure; the outlet of the runner (23) is detachably provided with a sampling head (24), and the sampling head (24) is provided with bristles (24 a) for sampling.
  3. 3. A pretreatment method according to claim 2, characterized in that said separate chamber further comprises a second separate chamber (24 b) recessed in the sampling head (24), the opening of the second separate chamber (24 b) being closed by the connection of the sampling head (24) to the flow channel (23) and being opened by the separation of the sampling head (24) from the flow channel (23); the connecting part (21) is provided with a rubber head (22), the first independent cavity is formed in the rubber head (22), a valve rod (25) is arranged in the runner in a sliding mode, one end of the valve rod (22) extends into the first independent cavity, the diaphragm (23) is annularly arranged between the runner (23) and the valve rod (25), and the valve rod (25) is used for receiving pressure from the outside of the rubber head (22) and descending and enabling the diaphragm (26) to be changed from a closed position to an open position and enabling the sampling head (24) to be separated from the runner (23).
  4. 4. A pretreatment method according to claim 3, wherein the fixative is preloaded into the sample fixation chamber or the first independent chamber, and the diluent is preloaded into at least one of the first independent chamber and the second independent chamber; The nucleic acid protecting agent is in a fixing solution or a diluting solution or is independently in a second independent chamber.
  5. 5. The pretreatment method of claim 2, wherein the fixative, the diluent, and the nucleic acid protecting agent are preloaded in a first separate chamber; Or the fixing liquid is preloaded in the sample fixing cavity, the diluent is preloaded in the first independent cavity, and at least one of the fixing liquid and the diluent contains a nucleic acid protecting agent.
  6. 6. The pretreatment method according to claim 1, wherein the sampling tube comprises a tube body (1) and a cover body (2), and the cover body (2) is provided with a connecting part (21) detachably connected with the tube body (1); The sample fixing cavity is arranged in the tube body; the cover body (2) further comprises a rubber head (22) and a shaft sleeve which are respectively arranged at two ends of the connecting part (21), the bottom end of the shaft sleeve is detachably provided with a sampling head (24), a valve rod (25) is slidably arranged in the shaft sleeve, and one end of the valve rod (25) extends into the rubber head (22); The independent chamber comprises a second independent chamber (24 b) concavely arranged on the sampling head (24), and an opening of the second independent chamber (24 b) is closed due to the connection of the sampling head (24) and the shaft sleeve and is opened due to the separation of the sampling head (24) and the shaft sleeve; the valve rod (25) is used for receiving pressure from outside the rubber head (22) and descending and separating the sampling head (24) from the shaft sleeve; the fixing liquid is preloaded in the sample fixing cavity, and the diluent is preloaded in the second independent cavity.
  7. 7. The pretreatment method according to any one of claims 1 to 6, wherein the diluent is nuclease-free water; The nucleic acid protecting agent is at least one selected from nuclease inhibitor, pH stabilizer and metal chelating agent; the pH stabilizer is at least one selected from HEPES, tris-HCl and PBS; The metal chelating agent is at least one selected from EDTA, EGTA and DTPA.
  8. 8. The pretreatment method of claim 7, wherein the nucleic acid protecting agent comprises a nuclease inhibitor, HEPES buffer, and EDTA; In the step S3, after the fixing solution and the diluting solution are mixed, the concentration of the nuclease inhibitor in the mixed solution is 40-200U/mL, the concentration of the HEPES buffer solution is 10-20 mM, and the concentration of EDTA is 1-5 mM.
  9. 9. The pretreatment method according to any one of claims 1 to 6, wherein in step S3, when the diluent is mixed with the fixative before or simultaneously with the occurrence of step S2, the concentration of the fixative in the mixed solution is 0.25 to 1%; When the subsequent mixing of the diluent with the fixative occurs at step S2, the concentration of the fixative in the mixed liquor does not exceed 1%; the fixing agent is selected from paraformaldehyde or formaldehyde.
  10. 10. The pretreatment method of claim 9, wherein in step S3, when the diluent is mixed with the fixative solution before or simultaneously with the occurrence of step S2, the concentration of the fixative in the mixed solution is 0.25 to 0.5%.
  11. 11. The pretreatment method as claimed in claim 1, further comprising the steps of: S4, directly transferring the closed sampling tube into a low-temperature freezing storage and enabling the sampling tube to undergo 1-3 cycles of freezing and thawing cycle; S5, opening a sampling tube, preparing a sample into a microbial single-cell suspension, and adding lysozyme into the microbial single-cell suspension to permeabilize microbial cell walls.
  12. 12. The pretreatment method of claim 11, wherein in step S4, the freezing temperature is between-80 ℃ and-16 ℃; In the step S5, the final concentration of lysozyme in the system is 50,000-100,000U/mL; In step S5, after lysozyme is added, the sampling tube is placed at 30-37 ℃ for incubation of 5-15 min.
  13. 13. A method for sequencing RNA from a single cell of a microorganism, comprising: processing a sample using the pretreatment method of any one of claims 1-12; And carrying out in-situ reverse transcription and library establishment by taking the pretreated microbial single cell body as a micro-reaction system, thereby obtaining single bacterial RNA sequencing data.

Description

Pretreatment method of microbial single-cell sequencing sample and microbial single-cell RNA sequencing method Technical Field The invention belongs to the technical field of biology, and particularly relates to a pretreatment method of a microbial single-cell sequencing sample and a microbial single-cell RNA sequencing method. Background Microbial single cell sequencing is a type of high resolution analysis technique that can resolve the characteristics and functional heterogeneity of individual microbial molecules in complex microbial communities or specific cultured strains. Existing single bacterial RNA sequencing systems (e.g., smRandom-seq, bacDrop, etc.) generally include 1) sample immobilization, 2) cell wall permeabilization, and 3) in situ reverse transcription and pool sequencing, the overall flow is shown in FIG. 19. In step 1), the conventional method relies on chemical immobilization of 4% (w/v, i.e., 4g/100 mL) Paraformaldehyde (PFA) overnight in a4℃shaker. During immobilization, PFA maintains cellular structure and nucleic acid status by cross-linking RNA, DNA, and proteins. To avoid excessive cross-linking, it is necessary to centrifuge the fixative immediately after the end of fixation and to re-suspend the pellet with buffer. This process is typically performed in open centrifuge tubes, which require multiple uncapping or transferring fluids during operation. In step 2), the immobilized cells are moderately permeabilized by a surfactant (such as 0.04% Tween-20) and lysozyme to form a permeable channel, so that the reverse transcription system can enter the inside of the cells conveniently. In the step 3), the bacterial body is used as a micro-reaction system, RNA in-situ reverse transcription is realized through a random primer, a poly (dA) tail is added under the catalysis of TdT, single bacteria and microbeads are encapsulated together by utilizing microfluidic droplets, and a microbead-poly (T) primer is released through enzyme digestion, so that cDNA barcoding and UMI marking are completed. Finally, a sequencing library is constructed through demulsification, purification, amplification and linker connection. However, the prior art still has the following problems that in the fixing and preserving process of the sample, the reagent needs to be added or removed by uncovering for many times, so that the sample and the reagent are exposed to the air, the pollution risk is increased, the operation is complicated, and the method is difficult to be suitable for the closed environment and the limited operation condition of the space station. Disclosure of Invention The invention aims to provide a pretreatment method of a microbial single-cell sequencing sample and a microbial single-cell RNA sequencing method, wherein the pretreatment method can realize sample fixation and preservation in a closed environment (namely, the whole process does not need to be uncapped), is simple and convenient to operate, has low sample exposure risk and can effectively keep nucleic acid stability. In order to achieve the above object, the present invention has the following technical scheme: The invention provides a pretreatment method of a microbial single-cell sequencing sample, which is carried out in a sampling tube, wherein a sample fixing cavity for accommodating the sample and fixing the sample and at least one independent cavity isolated from the sample fixing cavity are arranged in the sampling tube, diluent is pre-arranged in the at least one independent cavity, the sample fixing cavity or the at least one independent cavity is pre-arranged with the diluent, and at least one of the diluent and the diluent contains nucleic acid protecting agent; the pretreatment method comprises the following steps: S1, sampling a sample into a sampling tube and closing the sampling tube; s2, mixing the sample with a fixing solution in the closed sampling tube simultaneously or subsequently to the step S1 to fix the cells; and S3, mixing the diluent with the fixing solution to dilute the fixing solution before, simultaneously with or after the step S2 in the closed sampling tube. In the pretreatment method, the sample fixing process is carried out in a special sampling tube, the sampling tube is only required to be opened when the sample is taken in the whole process, and after that, the whole fixing process is carried out in a closed environment without uncapping. The method is characterized in that all reagents required for fixation are preloaded into the sampling tube, the sampling tube can be immediately closed after a sample is taken, the uncapping time of the sampling tube is shortened to the greatest extent, the exposure risk of the sample and the reagents is reduced to the lowest, the diluent is preloaded into the sampling tube, the concentration of the fixative in a sample fixation system can be reduced by the diluent, the purpose that the fixative is not required to be removed after the fixation is finished