WO2023241721A1 - Topology capture-based rna sample treatment system - Google Patents

Topology capture-based rna sample treatment system Download PDF

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WO2023241721A1
WO2023241721A1 PCT/CN2023/101050 CN2023101050W WO2023241721A1 WO 2023241721 A1 WO2023241721 A1 WO 2023241721A1 CN 2023101050 W CN2023101050 W CN 2023101050W WO 2023241721 A1 WO2023241721 A1 WO 2023241721A1
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rna
locked
cdna
permeable membrane
selectively permeable
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PCT/CN2023/101050
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French (fr)
Chinese (zh)
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张经纬
刘铁民
李婷
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复旦大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/20—After-treatment of capsule walls, e.g. hardening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to a method for separating analytes (such as cells, bacteria, viruses, nucleic acids, biochemical compounds and/or other materials) and targeted capture of biomolecules in a reaction compartment surrounded by a selectively permeable membrane.
  • analytes such as cells, bacteria, viruses, nucleic acids, biochemical compounds and/or other materials
  • Biomolecules derived from the same analyte are enriched in a reaction compartment surrounded by a selectively permeable membrane through specific capture reagents.
  • the remaining biomolecules can freely pass through the selective permeation membrane, thereby achieving various reaction substance exchanges required for purification, cleaning or multi-step reactions.
  • the captured enriched biomolecules and their reactive derivatives can be released from the reaction compartment surrounded by a selectively permeable membrane at the desired step.
  • the methods disclosed herein illustrate the use of reaction compartments surrounded by selectively permeable membranes for genotypic or phenotypic analysis of single cells.
  • Genome-wide transcriptome analysis is widely used, and the early method was to obtain RNA from a large number of tissue samples for sequencing.
  • this traditional method relies on the overall analysis of gene expression of millions of cells at once, which often masks biologically meaningful gene expression differences in certain specialized cells in specific tissues.
  • certain cells are rare in number and no other technique can analyze them except single-cell methods.
  • Single-cell gene expression analysis overcomes these limitations and can be used to mine gene regulatory networks across the entire genome, especially for highly heterogeneous stem cells and cell populations in early embryonic development. Combined with live cell imaging systems, single-cell transcriptome analysis can help to gain a deeper understanding of processes such as cell differentiation, cell reprogramming, and transdifferentiation, as well as related gene regulatory networks. Applying this technology to clinical practice, it is theoretically possible to continuously track the dynamic changes in gene expression under physiological or pathological conditions, thereby monitoring the progression of the disease.
  • Another application area of single-cell transcriptome analysis is the discovery of gene expression profiles of subcellular components, such as transcriptome analysis of genes specifically expressed in the axonal or dendritic parts of neurons, which are often important for the biology of the cell. Functionality plays an important role.
  • the Smart-seq2 method uses oligo-dT, TSO, and ISPCR primers with fixed sequences for reverse transcription amplification, which can effectively improve the amplification efficiency of cDNA.
  • sequencing is performed using an Illumina high-throughput sequencer, for example, the proportion of valid data obtained from the high-throughput sequencing library constructed using this method is low, which will have an adverse impact on subsequent data analysis.
  • An RNA sample processing system based on topological capture which includes:
  • the selectively permeable membrane can selectively pass through the RNA sample processing reagent
  • RNA to be analyzed and the RNA capture reagent are connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are transformed through biological or chemical reactions.
  • the selectively permeable membrane can selectively retain the whole or complex or nucleic acid content formed by connecting the RNA capture reagent and the RNA to be analyzed;
  • the diameter of the whole or complex or nucleic acid content is greater than 1/2 the pore size of the membrane pores of the selectively permeable membrane.
  • RNA sample processing system based on topological capture according to item 1, wherein,
  • RNA capture reagent RNA to be analyzed located inside the reaction compartment, and the whole or complex formed by connecting the RNA capture reagent and the RNA to be analyzed, or the deoxyribonucleic acid (DNA) and/or the deoxyribonucleic acid (DNA) formed after the conversion or the nucleic acid content of ribonucleic acid (RNA) is liquid, gelatinous or semi-liquid;
  • the reaction compartment also contains an osmotic pressure regulator inside;
  • the osmotic pressure regulator is dextran.
  • RNA sample processing system based on topological capture according to any one of items 1 to 2, wherein,
  • the capture reagent is a colloidal polymer compound formed by the polymerization of an oligonucleotide primer with a double bond itself or a polymerization of an oligonucleotide primer with a double bond and one or more compound monomers with a double bond. Colloidal polymer compound formed;
  • the RNA capture reagent performs a reverse transcription reaction with the RNA to be analyzed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  • a PCR amplification reaction is performed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  • RNA sample processing system based on topological capture according to any one of items 1 to 4, wherein,
  • the RNA capture reagent and the RNA to be analyzed are connected into a whole or a complex, or the nucleic acid content of the deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after the conversion, Further capture is performed with the following second capture reagent:
  • the second capture reagent is a complex of DNA transposase and DNA
  • the DNA transposase is Tn5, and the DNA has a transposase recognition sequence at its end.
  • RNA sample processing system based on topological capture according to any one of items 1 to 5, wherein,
  • the RNA to be analyzed originates from the same cell or cell nucleus; preferably, the RNA to be analyzed is in a complete in a cell or nucleus.
  • preparing a first phase including a tonicity regulator and a first aqueous solvent
  • RNA capture reagent is added in the first or second phase
  • RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
  • the above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
  • a reaction compartment with a selectively permeable membrane on the outside and content on the inside is mixed into a cell lysate to release RNA in cells or nuclei or is heated to release RNA in cells or nuclei to be contacted with an RNA capture reagent.
  • RNA sample processing system based on topological capture which includes the following steps:
  • preparing a first phase including an RNA capture reagent, a tonicity regulator, and a first aqueous solvent;
  • RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
  • the cell lysate into the first phase or the second phase; preferably the cell lysate and the cells or cell nuclei are in different phases;
  • the above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
  • the water-in-oil emulsion that has been cured or semi-cured is demulsified to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the inside.
  • composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
  • cDNA first-strand complementary deoxyribonucleic acid
  • TSO template switching oligonucleotide
  • LNA locked nucleic acid
  • transposome comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample
  • the RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of items 1 to 6 connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after biological or chemical reaction with the RNA capture reagent,
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the template switching oligonucleotide comprises one or two ribonucleotide residues and zero or more locked nucleic acid (LNA) residues.
  • locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  • SEQ ID NO.1 AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
  • ii.rGrG+N preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
  • iv.rG+G+G preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  • cDNA is in a solution mixed with oligonucleotide primers or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
  • oligonucleotide primer comprises SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, where "N” is any nucleoside base, and "V” Is it “A” or “C” or “G”.
  • a method for analyzing gene expression in multiple single cells comprising the following steps: preparing a cDNA library according to the method of item 9; and sequencing the cDNA library.
  • TSO template switching oligonucleotide
  • RNA is RNA released from cleaving single cells in a reaction compartment surrounded by a selectively permeable membrane.
  • the single cell according to item 29 characterized in that the single cell is wrapped into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
  • transposon end domain comprises a Tn5 transposon end structure area.
  • a method for preparing a next-generation sequencing (NGS) library using ribonucleic acid (RNA) samples in a reaction compartment surrounded by a selectively permeable membrane comprising:
  • composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
  • cDNA first-strand complementary deoxyribonucleic acid
  • TSO template switching oligonucleotide
  • LNA locked nucleic acid
  • the RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of items 1 to 5 connected into a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after biological or chemical reaction with the RNA capture reagent,
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  • i.rGrG+G preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
  • ii.rGrG+N preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
  • iv.rG+G+G preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  • cDNA is in a solution mixed with an oligonucleotide primer or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
  • oligonucleotide primer comprises SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N” is any nucleobase and "V” is "A” or "C” or “G”.
  • a method for analyzing gene expression in multiple single cells comprising the steps of: preparing a cDNA library using the method according to item 36; and sequencing the cDNA library.
  • TSO template switching oligonucleotide
  • the template switching oligonucleotide according to item 52 which contains three nucleotide residues at the 3'-end, and the nucleotide residues are selected from +N+N+N, N+N+ N, NN+N, rN+N+N, and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Each occurrence of +N is independently a locked nucleotide residue.
  • the template switching oligonucleotide of item 52 wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  • RNA is RNA released by cleavage of single cells in a reaction compartment surrounded by a selectively permeable membrane.
  • the single cell according to item 56 characterized in that it is packaged into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
  • transposase comprises Tn5 transposase.
  • transposon terminus domain comprises a Tn5 transposon terminus domain.
  • the method of item 36 wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product cDNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
  • Microfluidic system is used to form a colloidal polymer composed of an oligonucleotide (primer) with a double bond and one or more compound monomers with a double bond and the selective permeation of cells.
  • a reaction compartment surrounded by a membrane.
  • FIG. 1B A microfluidic system is used to form a reaction compartment surrounded by a selectively permeable membrane surrounding cells.
  • the analyte is a DNA molecule
  • the capture reagent can be one or more of proteins, nucleic acids, and complexes formed by them together.
  • the analyte is an RNA/DNA hybrid chain molecule
  • the capture reagent can be one or more of proteins, nucleic acids, and complexes formed by them together.
  • the analyte is an RNA molecule
  • the capture reagent can be a colloidal polymer polymerized by an oligonucleotide (primer) with a double bond and one or more compound monomers with a double bond.
  • oligonucleotide primer
  • compound monomers monomers with a double bond.
  • the analyte is an RNA molecule
  • the capture reagent can be an oligonucleotide (primer).
  • FIG. 1 Microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
  • the first fluid (phase I solution, rich in dextran) and the second fluid (phase II solution, rich in polyethylene glycol-based polymer) are injected into the microfluidic chip through the microfluidic device system.
  • continuous phase the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer); target capture reagent Enter the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.
  • Figure 10 mRNA capture microspheres and cells in the reaction compartment; microspheres ⁇ 2.7 microns; outer wall pore size ⁇ 50nm.
  • Cell lysis occurs in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 13 Removal of genomic DNA occurs in a reaction compartment surrounded by a selective permeable membrane.
  • Figure 14 Fluorescent staining of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 15 Alkali-dissolved agarose gel for single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 16 Qsep characterization of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 18 cDNA amplification library fragmentation in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 19 Qsep characterization of cDNA amplification library interruption in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 20 Interruption of RNA/DNA hybrid strands in a reaction compartment surrounded by a selectively permeable membrane.
  • FIG. 21 Bioanalzyer 2100 characterization of RNA/DNA hybrid chain disruption in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 22 Before gelation of a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
  • Figure 23 After gelation of the reaction compartment consisting of a selectively permeable membrane (magnetic bead topological capture).
  • FIG. 24 After demulsification of reaction compartments composed of selectively permeable membranes (magnetic bead topological capture).
  • FIG. 25 Cell lysis in a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
  • Figure 26 Removal of genomic DNA (magnetic bead topological capture) in a reaction compartment composed of a selectively permeable membrane.
  • Figure 27 Fluorescent staining of single-cell mRNA reverse-transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
  • substantially free with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the specific component is present in an amount undetectable by standard analytical methods.
  • the present application provides an RNA sample processing system based on topological capture.
  • a topological capture-based RNA sample processing system which includes:
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the whole body or complex or nucleic acid content; the diameter of the whole body or complex or nucleic acid content is greater than 1/2 of the pore diameter of the membrane pores of the selectively permeable membrane.
  • the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
  • reaction compartment means a semi-enclosed reaction space surrounded by a semi-permeable membrane that allows the reaction of some substances (depending on their molecular size, hydrophobicity/hydrophilicity) Different, carry different charges, etc.) exchange, specifically for this application, molecules with a smaller pore size relative to the semipermeable membrane can freely enter or exit the reaction compartment, relative to the larger pore size of the semipermeable membrane molecules can be trapped within the reaction compartment.
  • nucleic acid specifically refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • the English equivalent of “diameter” is equivalent diameter, more specifically, it can be understood as “Stokes diameter", which means when the particle under study has the same final settling velocity as a certain spheroidal body , the diameter of the spheroids is the Stokes diameter of the particles under study.
  • the above-mentioned RNA sample processing system based on topological capture wherein the RNA capture reagent and the RNA to be analyzed are located inside the reaction compartment, and the RNA capture reagent and the RNA to be analyzed are connected and The whole or complex formed, or the deoxyribonucleic acid (DNA) and/or nuclei formed after said transformation
  • the nucleic acid content of RNA is in a liquid, colloidal or semi-liquid state; the inside of the reaction compartment also contains an osmotic pressure regulator; preferably, the osmotic pressure regulator is dextran.
  • topology capture is a professional term in this field, and “topology” corresponds to the English topology.
  • Topological trapping is a new concept combining supramolecular chemistry and topology: a mechanical interlocking structure based on non-covalent bonding, composed of a combination of selectively permeable membranes and particles, used to target and retain biomolecules.
  • a “mesh-shaped" semi-permeable membrane structure is formed, which has a clear interception effect on molecules with large diameters (large diameters generally correspond to molecules with large molecular weight, but it is also related to the shape of the molecules). This interception can be adjusted by the pore size of the "mesh-shaped semipermeable membrane”.
  • the above-mentioned RNA sample processing system based on topological capture is provided, wherein the capture reagent is a colloidal polymer compound formed by self-polymerization of an oligonucleotide primer with a double bond or a polymer with a double bond.
  • the capture reagent is a colloidal polymer compound formed by self-polymerization of an oligonucleotide primer with a double bond or a polymer with a double bond.
  • the RNA capture reagent performs a reverse transcription reaction with the RNA to be analyzed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  • the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
  • oligonucleotide primer with double bonds specifically refers to a “dual-action primer” that not only has the function of a general PCR primer, but also has the function of "forming larger-diameter molecules through condensation, and then using Acts as a capture reagent for the RNA to be analyzed.
  • dual-action primer that not only has the function of a general PCR primer, but also has the function of "forming larger-diameter molecules through condensation, and then using Acts as a capture reagent for the RNA to be analyzed.
  • a PCR amplification reaction is performed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  • the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
  • RNA to be analyzed specifically refers to mRNA, which can undergo a reverse transcription reaction under the action of reverse transcriptase, appropriate solvent, and temperature to generate cDNA, thereby forming an mRNA/cDNA hybrid chain.
  • the reverse transcription reaction can be performed in a reverse transcription PCR instrument.
  • PCR is the English abbreviation of "polymerase chain reaction”. PCR uses DNA to denature into a single strand at a high temperature of 95°C in vitro. At low temperature (usually around 60°C), the primers are combined with the single strand according to the principle of base pairing, and then the temperature is adjusted to the optimal reaction of the DNA polymerase.
  • DNA polymerase synthesizes complementary strands along the direction from phosphate to five-carbon sugar (5'-3').
  • the PCR machine based on polymerase is actually a temperature control device, which can adjust the temperature at denaturation temperature, renaturation temperature, and extension temperature. Control accurately between degrees.
  • the above-mentioned topological capture-based RNA sample processing system is provided, wherein,
  • the RNA capture reagent and the RNA to be analyzed are connected into a whole or a complex, or the nucleic acid content of the deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after the conversion, Capture is further performed by the following second capture reagent: the second capture reagent is a complex of DNA transposase and DNA; preferably, the DNA transposase is Tn5, and the DNA has transposase recognition at its end. sequence, specifically a transposase with a 19 bp ME sequence at both ends.
  • Tn5 transposase can serve as a second capture reagent (to form a larger diameter molecule to be trapped) is that the 3'-OH of Tn5 nucleophilically attacks the target sequence, forming a 9 bp gap between the transposon insertion sites. At the sticky end, a covalent bond is formed between the 3'-OH of the transposon and the 5'-P of the target DNA. Based on the affinity of its dimer protein interaction, Tn5 forms a complex with the fragmented short DNA fragments, maintaining the order and contiguity of the original DNA molecules. )
  • an RNA sample processing system based on topological capture wherein the RNA to be analyzed originates from the same cell or nucleus; preferably, the RNA to be analyzed is in a complete cell or nucleus.
  • the RNA to be analyzed is first in intact cells or nuclei, then undergoes a cell lysis stage, and is in a state of being released from the cells/nuclei when captured by the capture reagent.
  • the present application relates to a method of manufacturing the above-mentioned topological capture-based RNA sample processing system.
  • a method for manufacturing the above-mentioned topological capture-based RNA sample processing system includes the following steps: preparing a first phase including a tonicity regulator and a first aqueous solvent; preparing a mixture of selective permeability a second phase of film-forming material and a second aqueous solvent;
  • the RNA capture reagent is added to the first phase or the second phase; the RNA to be analyzed in intact cells or cell nuclei is mixed into the first phase or the second phase; preferably the RNA to be analyzed in intact cells or cell nuclei is mixed to the first phase; mix the first phase and the second phase into a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and conduct the above-mentioned water-in-oil emulsion.
  • the curing or semi-curing reaction forms a selectively permeable membrane;
  • the reaction compartment with its contents inside is mixed into a cell lysis solution to release RNA within the cells or nuclei or is heated to release RNA within the cells or nuclei into contact with the RNA capture reagent.
  • the first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , potassium phosphate, glucose, other none Organic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl cellulose, ethylene oxide- Propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);
  • the second aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber Element, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);
  • both the first aqueous solvent and the second aqueous solvent are water;
  • the osmotic pressure regulator is dextran and the concentration of dextran in the first aqueous solvent is 3%-10%; most preferably the osmotic pressure regulator is dextran and the dextran The concentration of sugar in the first aqueous solvent is 5.5%;
  • oil phase solvent is selected from one or more of the following groups: perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer, HFE-7500 fluorinated oil, Squalane oil, silicone oil and mineral oil;
  • the oil phase solvent is a perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer.
  • the adjustment conditions for the curing or semi-curing reaction are the illumination intensity and illumination time of ultraviolet light
  • the catalyst used in the curing or semi-curing reaction is one or more TEMED initiators selected from the following group: 2-hydroxy-2-methyl-1-phenyl acetone, 1-hydroxycyclohexylphenylmethyl Ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2,4,6-trimethylbenzoyl-diphenyl Phosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl]- 1-Butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, MBF methyl benzoylformate, benzoin and derivatives ( Benzoin, benzoin dimethyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin but
  • the selectively permeable membrane forming material is selected from one or more of the following group: polyolefin, olefin copolymer, acrylic, vinyl polymer, polyester, polycarbonate, polyamide, polyimide, Formaldehyde resin, polyurethane, ether polymer, cellulose, thermoplastic elastomer and thermoplastic polyurethane materials;
  • the selectively permeable membrane-forming material is a hydrogel framework material; further preferably, the selectively permeable membrane-forming material is polyethylene glycol diacrylate (PEGDA), and further preferably, the PEGDA is in a second aqueous solvent.
  • PEGDA polyethylene glycol diacrylate
  • concentrated Degree is 1%-10%
  • PEGDA PEGDA and the concentration of PEGDA in the second aqueous solvent is 3%.
  • demulsification can be achieved by shaking the mixture with surfactants followed by high-speed centrifugation.
  • a method for manufacturing the above topological capture-based RNA sample processing system includes the following steps: preparing a first phase including an RNA capture reagent, an osmotic pressure regulator and a first aqueous solvent; preparing to mix A second phase of selectively permeable membrane-forming material and a second aqueous solvent; mixing the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mixing the RNA in intact cells or cell nuclei The RNA to be analyzed is mixed into the first phase;
  • the cell lysate into the first phase or the second phase; preferably the cell lysate and the cells or cell nuclei are in different phases; mix the first phase and the second phase into a mixed hydrophilic phase, and then mix the mixed hydrophilic phase Mix with the oily solvent to prepare a water-in-oil emulsion; and the cell lysate assists in releasing the RNA in the cells or nuclei to contact the RNA capture reagent; and the above-mentioned water-in-oil emulsion is cured or semi-cured to form a selectively permeable emulsion. Passing the membrane; demulsifying the water-in-oil emulsion that has been cured or semi-cured to obtain a reaction compartment with a selectively permeable membrane on the outer layer and content inside.
  • the present application relates to a method for preparing a next-generation sequencing (NGS) library based on a topological capture-based RNA sample processing system.
  • NGS next-generation sequencing
  • a method for preparing a next-generation sequencing (NGS) library based on an RNA sample processing system based on topological capture includes: (a) preparing a composition including: RNA sample; first strand complementary deoxyribonucleic acid (cDNA) primers; template switch oligonucleotides; reverse transcriptase; and dNTPs; annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and by making the RNA -
  • the cDNA intermediate is contacted with a template switching oligonucleotide (TSO) to perform the reverse transcriptase reaction, wherein the TSO contains or does not contain a locking nucleic acid (LNA) at its 3'-end, in a position suitable for the first cDNA strand Under extension conditions, it is complementary to the RNA molecule, making it complementary to TSO; (b) Under amplification conditions sufficient
  • the RNA sample is the above-mentioned RNA to be analyzed and the RNA capture reagent connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are biologically or A chemical reaction that produces deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) that is transformed
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • second-generation sequencing is to distinguish it from the first-generation DNA sequencing technology that came out in the 1970s, so it is also called “next-generation sequencing”; because it can simultaneously analyze millions or even hundreds of Billions of different DNA fragments are sequenced, and this technology is also called “high-throughput sequencing” (the nucleic acid "interruption” step in this application is to prepare for high-throughput sequencing).
  • Next-generation sequencing has reduced the cost of DNA sequencing by at least five orders of magnitude, making it widely used in various fields of biology.
  • second-generation sequencing is usually divided into two categories: sequencing by synthesis and sequencing by ligation.
  • the second-generation sequencing in this application is based on sequencing by synthesis.
  • LNA is the English abbreviation of locked nucleic acid, which is called “locked nucleic acid” or “locked nucleic acid” in Chinese.
  • LNA can combine with DNA or RNA to form hybrid oligonucleotide molecules. Therefore, the introduction of LNA can improve various technologies based on hybridization principles such as PCR reactions, microarray chips, and in situ hybridization. This type of nucleic acid can increase the melting problem of primers or probes, enhance the stability of these substances, and can be used in technologies such as real-time fluorescence quantitative PCR.
  • cDNA is a cloned collection of DNA (complementary DNA) that is complementary to cellular messenger RNA (i.e., mRNA).
  • TSO messenger RNA
  • Template switch oligo which is a template switch oligonucleotide. It is an oligonucleotide.
  • the C oligonucleotide is added to the 5-end of the non-template strand by reverse transcriptase for Downstream cDNA amplification.
  • MMLV reverse transcriptase adds some additional nucleotides (mainly deoxycytosine) to the 3' end of the newly synthesized cDNA strand. glycosides).
  • TSO is used to cooperate with reverse transcriptase to initiate reverse transcription of the transcriptome.
  • methyl donor refers to a related compound that donates a methyl group (-CH3) to the acceptor during the reaction.
  • the above method is provided, wherein the methyl donor is betaine.
  • the above method is provided, wherein the metal salt is a magnesium salt.
  • the above method is provided, wherein the template switching oligonucleotide comprises one or two ribonucleotide residues and zero or more locked nucleic acid (LNA) residues.
  • LNA locked nucleic acid
  • the above method is provided, wherein the one or two ribonucleotide residues are riboguanine.
  • the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  • the method above is provided, wherein the locked nucleic acid residue is locked guanine.
  • the above method is provided, wherein the template switching oligonucleotide comprises three nucleotide residues at the 3'-end characterized by the molecular formula rGrG+N, where +N represents locked Nucleotide residues.
  • the above method is provided, wherein the template switching oligonucleotide comprises rGrG+G.
  • the template switching oligonucleotide is selected from the group consisting of:
  • i.rGrG+G preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
  • ii.rGrG+N preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
  • iv.rG+G+G preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  • rG and rC refer to G and C respectively as ribonucleic acid.
  • the above method is provided, wherein the cDNA is in a solution mixed with an oligonucleotide primer or in a It is synthesized from a colloidal polymer compound formed by polymerizing an oligonucleotide primer with a double bond and one or more compound monomers with a double bond.
  • the above method is provided, wherein the oligonucleotide primer comprises SEQ ID NO. 5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNN NNNN, wherein "N” is any nucleoside base, and “V” is "A” or "C” or “G”.
  • the present application provides methods for analyzing gene expression in multiple single cells.
  • the above method includes the following steps: preparing a cDNA library according to the above method; and sequencing the cDNA library.
  • the present application provides a template switching oligonucleotide (TSO).
  • TSO template switching oligonucleotide
  • a TSO as described above which contains a locked nucleotide residue at its 3'-end.
  • TSO wherein it comprises three nucleotide residues at the 3'-end, the nucleotide residues being selected from +N+N+N, N+N+N , NN+N, rN+N+N and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Occurrences of +N are independently locked nucleotide residues.
  • the above-described TSO is provided, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine, and locked of 5-methylcytosine.
  • TSO as described above, wherein the three nucleotide residues are selected from the group consisting of NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
  • the above-mentioned library construction method by second-generation sequencing is provided, wherein the RNA is RNA released from cleavage of single cells in a reaction compartment surrounded by a selectively permeable membrane.
  • the above method is provided, wherein the single cell is packaged into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
  • the above method is provided, wherein the transposase is a Tn5 transposase.
  • the transposon terminus domain comprises a Tn5 transposon terminus domain.
  • the above method further comprises combining the first double-stranded product cDNA with the second double-stranded product DNA to generate a pooled cDNA sample, and then labeling the pooled cDNA sample.
  • the above method further comprises quantifying one or more RNA species of the RNA sample.
  • the above method is provided, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
  • a library construction method by second-generation sequencing includes: (a) formulating a composition including the following: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switching oligonucleotides; reverse transcriptase; and dNTPs; annealing a cDNA synthesis primer to an RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate; and converting an oligonucleotide by aligning the RNA-cDNA intermediate with a template
  • the reverse transcriptase reaction is carried out by contacting with acid (TSO), where the TSO contains or does not contain a locked nucleic acid (LNA) at its 3'-end, which is complementary to the RNA molecule under conditions suitable for the elongation of the first DNA strand, making it Complementary to TSO;
  • TSO acid
  • LNA locked nucleic acid
  • the RNA sample is the RNA to be analyzed and the RNA capture reagent mentioned above connected into a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA
  • the capture reagent undergoes a biological or chemical reaction to generate the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after conversion, wherein (a)-(c) are performed in the reaction compartment referred to above.
  • the above method is provided, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
  • the above method is provided, wherein the methyl donor is betaine.
  • the above method is provided, wherein the metal salt is a magnesium salt.
  • the above method is provided, wherein the magnesium salt has a concentration of at least 7mM.
  • said template switching oligonucleotide comprises one or two ribonucleotide residues and said zero or more locked nucleic acid (LNA) residues.
  • the above-described method is provided, wherein the one or two ribonucleotide residues are riboguanine.
  • the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  • the method above is provided, wherein the locked nucleic acid residue is locked guanine.
  • the above method is provided, wherein the template switching oligonucleotide comprises three nucleotide residues at the 3'-end characterized by the formula rGrG+N, where +N represents locked Nucleotide residues.
  • the above method is provided, wherein the template switching oligonucleotide comprises rGrG+G.
  • the above method is provided, wherein the methyl donor is betaine and the metal salt is MgCl2 at a concentration of at least 9mM.
  • the above method is provided, wherein the template switching oligonucleotide is selected from the group consisting of:
  • i.rGrG+G preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
  • ii.rGrG+N preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
  • iii.+G+G+G preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  • the above method is provided, wherein the cDNA is in a solution mixed with an oligonucleotide primer or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
  • the above method is provided, wherein the oligonucleotide primer comprises SEQ ID NO. 5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N” is any nucleobase, and “V” is “A” or “C” or “G”. "T30” stands for 30 T's.
  • the above method includes the following steps: preparing a cDNA library using the above method; and sequencing the cDNA library.
  • the above method is provided, wherein a locked nucleotide residue is included at its 3'-most end.
  • the above method is provided, wherein the RNA is RNA released by cleavage in a single cell in a reaction compartment surrounded by a selectively permeable membrane.
  • the above method is provided, wherein the microfluidic system is wrapped into a reaction compartment surrounded by a selectively permeable membrane.
  • the transposase comprises Tn5 transposase.
  • the transposon terminus domain comprises a Tn5 transposon terminus domain.
  • the above method further comprises combining the first double-stranded product cDNA with the second double-stranded product cDNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
  • the above method further comprises quantifying one or more RNA species of the RNA sample.
  • the method described in the above application is provided, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
  • aqueous two-phase system aqueous two-phase system
  • target capture reagents All chemicals were ordered from Sigma-Aldrich and Fisher Scientific.
  • APS ammonium persulfate
  • 10% (w/v) dextran MW 500K
  • 5'Acrydite poly T primer oligonucleotide primer with double bonds
  • SEQ ID NO.6 5'Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT ), 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) to prepare two-phase aqueous system droplets (hereinafter referred to as ATPS droplets) .
  • ATPS droplets two-phase aqueous system droplets
  • phase I solution i.e. phase I solution
  • phase II solution i.e. phase II solution
  • Emulsification see Figures 3 and 4
  • the reaction compartment surrounded by droplets and selectively permeable membranes is made using a height of 50 ⁇ m and a width of 40 ⁇ m.
  • the nozzle is produced by a microfluidic chip (see Figures 3 and 4).
  • Typical flow rates used are: a phase rich in PEGD(M)A and 5'Acrytite poly T primers (Phase I solution), a flow rate of 200 ⁇ L/h, a mixture rich in dextran and cells (Phase II solution), The flow rate was 100 ⁇ L/h and the droplet stabilizing oil (carrier oil) (2% PEG-PFPE 2 HFE7500), the flow rate was 600 ⁇ L/h.
  • carrier oil 2% PEG-PFPE 2 HFE7500
  • Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 ⁇ g/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA.
  • the reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 min and then at 50°C for an additional 30 min (as shown in Figure 12, showing the post-lysis state ).
  • RNA is captured by the capture reagent (hardened 5'Acrydite poly T primer) and remains in the reaction compartment ( Figure 2C and Figure 10 respectively show the hardened bands captured by mRNA in the reaction compartment).
  • the reaction compartment surrounded by a selective permeable membrane is used to remove rRNA and genomic DNA ( Figure 13).
  • Two kits are provided, namely MGIEasy rRNA Removal Kit (Cat. No.: MGI, 1000005953). DNase I (Cat.
  • RNA reverse transcription and amplification Suspend the reaction compartment surrounded by the selectively permeable membrane obtained in the previous step in reverse transcriptase containing 0.5U/ ⁇ L Superscript IV and 1X First Strand Buffer, and then incubate at 50°C for another 30 minutes.
  • PCR which is similar to a macro reaction, is used to amplify fragments of specific regions of cDNA or specific genes. Each amplification was performed for 35 cycles using KAPA PCR kit (KAPA Biosystems, KK2602). In all enzymatic reactions, the reaction compartment surrounded by the selectively permeable membrane occupies approximately 40-50% of the final reaction volume ( Figure 14, Figure 15, Figure 16 and Figure 17).
  • Figure 14 shows fluorescent staining of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane
  • Figure 15 shows single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane. Results
  • Figure 16 shows Qsep characterization of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane
  • Figure 17 Single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane , for flow analysis and sorting.
  • Example 2 Single cell mRNA capture (interruption of RNA/DNA hybrid chain, that is, no PCR amplification, direct reverse transcription and then interruption)
  • Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
  • ATPS droplets Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 5'Acrytide poly T primer: For example, SEQ ID NO. 6: 5'Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) can be used to prepare ATPS liquid droplets (hereinafter referred to as ATPS droplets).
  • ATPS droplets ATPS liquid droplets
  • phase II solution concentration of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used.
  • the solutions containing all the above components are mixed and liquid-liquid phase separation is induced in a tabletop centrifuge to obtain the upper phase, i.e., phase I solution, and the lower phase, i.e., phase II solution.
  • Figure 4 shows the injection of the first fluid (Phase I solution, rich in dextran), the second fluid (Phase II solution, rich in polyethylene-based) into the microfluidic chip through the microfluidic device system.
  • the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer) ;
  • PFPE-PEG-PFPE perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer
  • the target capture reagent enters the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.
  • FIG. 5 shows a method for hardening the outer layer II phase by initiating polymerization.
  • Figure 8 shows the reaction compartment surrounded by the selectively permeable membrane after gelation.
  • Figure 9 shows a reaction compartment surrounded by a selectively permeable membrane after demulsification.
  • Lysis of cells removal of rRNA and genomic DNA. Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 ⁇ g/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA.
  • the reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 minutes and then at 50°C for an additional 30 minutes (Figure 12, showing the post-lysis state).
  • FIG. 12 shows cell lysis in a reaction compartment surrounded by a selectively permeable membrane.
  • RNA is captured by the capture reagent (Acrytide polyT primer) and remains in the reaction compartment ( Figure 2C, Figure 10. Double-bonded oligonucleotides (primers and cells) captured by mRNA in the reaction compartment.
  • rRNA removal and genomic DNA removal are performed in a reaction compartment surrounded by a selectively permeable membrane ( Figure 13).
  • Figure 13 shows that genomic DNA is removed in a reaction compartment surrounded by a selectively permeable membrane.
  • FIG. 20 shows the interruption of RNA/DNA hybrid chains in a reaction compartment surrounded by a selectively permeable membrane.
  • Figure 21 shows the Bioanalzyer 2100 characterization of RNA/DNA hybrid chain interruption in a reaction compartment surrounded by a selective permeable membrane.
  • Example 3 Single-cell mRNA capture (magnetic bead topological capture, breaking DNA double strands)
  • Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
  • Streptavidin magnetic beads coupled 5' biotinylated polyT primer was performed by suspending magnetic beads in buffer containing: 10mM Tris-HCl (pH 7.5), 1mM EDTA, 1 M NaCl, 0.05% Tween-20, 1uM 5'biotinylated polyT. Place it on a rotating mixer and rotate and mix at room temperature for 30 minutes to prepare the capture reagent.
  • FIG. 4 For the generation of a reaction compartment surrounded by a selectively permeable membrane of larger size, as shown in Figure 1: the reaction compartment surrounded by droplets and selectively permeable membranes uses a 50 ⁇ m height and 40 ⁇ m wide nozzle Produced by microfluidic chips. Typical flow rates used are: Phase I solution rich in PEGD(M)A - flow rate 200 ⁇ L/h, Phase II solution rich in dextran, cells, capture reagent - flow rate 100 ⁇ L/h and droplet stabilizing oil ( 2% PEG-PFPE 2 HFE7500) - flow rate 600 ⁇ L/h.
  • Figure 4 shows the injection of the first fluid (Phase I solution, rich in dextran), the second fluid (Phase II solution, rich in polyethylene-based) into the microfluidic chip through the microfluidic device system.
  • the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer)
  • PFPE-PEG-PFPE perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer
  • Figure 22 shows the state of the reaction compartment composed of a selectively permeable membrane before gelation (magnetic bead topological capture).
  • FIG. 5 shows a method for hardening the outer layer II phase by initiating polymerization.
  • Figure 23 shows the state of the reaction compartment composed of a selectively permeable membrane after gelation (magnetic bead topological capture).
  • Figure 24 shows a reaction compartment composed of a selectively permeable membrane after demulsification (magnetic bead topological capture).
  • Lysis of cells removal of rRNA and genomic DNA. Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 ⁇ g/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA.
  • the reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 minutes and then at 50°C for an additional 30 minutes (Figure 25).
  • Figure 25 shows cell lysis (magnetic bead topological capture) in a reaction compartment consisting of a selectively permeable membrane.
  • RNA is captured by streptavidin magnetic beads and remains in the reaction compartment.
  • rRNA removal and genomic DNA removal are performed in a reaction compartment surrounded by a selectively permeable membrane ( Figure 26).
  • removal of genomic DNA is performed within a reaction compartment consisting of a selectively permeable membrane shown in Figure 26 .
  • the amplified products were fragmented using the DNA fragmentation kit (Nextera XT DNA Library PreparationKit (24samples), FC-131-1024) according to the manufacturer's recommendations.
  • Tn5 dimers as capture reagents can maintain short fragments of DNA in a polymeric state in a reaction compartment surrounded by a selectively permeable membrane.

Abstract

The present application discloses a cDNA synthesis method having improved high-throughput reverse transcription, template conversion and pre-amplification, so as to increase the yield and average length of cDNA libraries generated from single cells or single cell nuclei by means of high throughput. In the method and system, a large number of partitions (for example, a selective permeable compartment) are formed for performing single-cell or single-cell nucleus encapsulation, and the method and system allow single-operation or multi-operation chemical and/or biochemical treatment to be implemented within the partitions. Steps such as reverse transcription or full-length cDNA pre-amplification are performed within reaction compartment partitions, and fragmentation of a transposase complex-mediated full-length transcriptome amplification product is performed in each partition. This process retains library intermediate fragmented template DNA from same cells or cell nuclei in the selective permeable compartment. In addition, a partition (for example, a droplet) is secondarily formed by means of droplet microfluidics, and the selective permeable compartment and barcoded oligonucleotide microspheres of a fragmented full-length transcriptome amplification product from a single cell are encapsulated in the droplet, and the fragmented full-length transcriptome is labelled by means of the barcodes on the oligonucleotide microspheres.

Description

基于拓扑捕获的RNA样本处理系统RNA sample processing system based on topological capture 技术领域Technical field
本发明涉及一种在由选择性透过膜包围而成的反应隔室中分离待分析物(例如：细胞、细菌、病毒、核酸、生化化合物和/或其他材料)及靶向捕获生物分子的系统和方法，并通过反应/流程处理封装的生物分子以执行多步骤反应。来源于同一待分析物的生物分子通过特异性的捕获试剂富集在由选择性透过膜包围而成的反应隔室中。其余生物分子可自由通过选择性透过膜，从而实现纯化、清洗或多步反应所需要的各种反应物质交换。在用外部刺激处理后，捕获富集的生物分子及其反应的衍生物，可以在所需的步骤从选择性透过膜包围而成的反应隔室中释放出来。本发明揭示的方法举例说明了使用选择性透过膜包围而成的反应隔室对单个细胞进行基因型或表型分析。The present invention relates to a method for separating analytes (such as cells, bacteria, viruses, nucleic acids, biochemical compounds and/or other materials) and targeted capture of biomolecules in a reaction compartment surrounded by a selectively permeable membrane. Systems and methods and processing encapsulated biomolecules through reactions/processes to perform multi-step reactions. Biomolecules derived from the same analyte are enriched in a reaction compartment surrounded by a selectively permeable membrane through specific capture reagents. The remaining biomolecules can freely pass through the selective permeation membrane, thereby achieving various reaction substance exchanges required for purification, cleaning or multi-step reactions. After treatment with external stimuli, the captured enriched biomolecules and their reactive derivatives can be released from the reaction compartment surrounded by a selectively permeable membrane at the desired step. The methods disclosed herein illustrate the use of reaction compartments surrounded by selectively permeable membranes for genotypic or phenotypic analysis of single cells.
背景技术Background technique
全基因组范围内的转录组分析被广泛应用，早期的方法是从大量的组织样品中获取RNA进行测序。但此传统方法依赖于一次性总体分析数百万个细胞的基因表达，往往会掩盖特异组织中某些特化细胞具有生物学意义的基因表达差异。并且，在健康组织或肿瘤等病变组织中，某些细胞的数量稀少，除了单细胞方法没有任何其他的技术能够对它们进行分析。Genome-wide transcriptome analysis is widely used, and the early method was to obtain RNA from a large number of tissue samples for sequencing. However, this traditional method relies on the overall analysis of gene expression of millions of cells at once, which often masks biologically meaningful gene expression differences in certain specialized cells in specific tissues. Moreover, in healthy tissues or diseased tissues such as tumors, certain cells are rare in number and no other technique can analyze them except single-cell methods.
单细胞基因表达分析则克服了这些局限，可用于在全基因组范围内挖掘基因调节网络，尤其适用于存在高度异质性的干细胞及胚胎发育早期的细胞群体。与活细胞成像系统相结合，单细胞转录组分析更有助于深入理解细胞分化、细胞重编程及转分化等过程及相关的基因调节网络。将此技术应用于临床，理论上可以在生理或病理情况下连续追踪基因表达的动力学变化，从而监测疾病的进展。单细胞转录组分析的另一应用领域是发现亚细胞成分的基因表达谱，例如对在神经元的轴突或树突部分特异表达的基因的转录组的分析，这些基因往往对细胞的生物学功能发挥着重要作用。Single-cell gene expression analysis overcomes these limitations and can be used to mine gene regulatory networks across the entire genome, especially for highly heterogeneous stem cells and cell populations in early embryonic development. Combined with live cell imaging systems, single-cell transcriptome analysis can help to gain a deeper understanding of processes such as cell differentiation, cell reprogramming, and transdifferentiation, as well as related gene regulatory networks. Applying this technology to clinical practice, it is theoretically possible to continuously track the dynamic changes in gene expression under physiological or pathological conditions, thereby monitoring the progression of the disease. Another application area of single-cell transcriptome analysis is the discovery of gene expression profiles of subcellular components, such as transcriptome analysis of genes specifically expressed in the axonal or dendritic parts of neurons, which are often important for the biology of the cell. Functionality plays an important role.
1990年，Norman Iscove的课题组首次证实对单细胞进行转录组分析是可行的，他们用PCR技术实现了对cDNA分子的指数级扩增。在20世纪90年代初期，Eberwine 等人发明了一种新技术，能够从单个的活神经元细胞中获得cDNA，并且再以这些cDNA为模板转录生成RNA，实现RNA的线性扩增。随着芯片时代的来临，科学家们用这些线性、和指数级扩增技术对单细胞之间的基因表达差异进行了大量的比较和研究。2008年时出现了高通量RNA测序技术，不久之后，这种技术与前面发展起来的核酸扩增技术结合起来，对单细胞转录组进行了更加精细的研究。2009年，英国剑桥大学Gurdon研究所Tang通过对单个小鼠卵裂细胞(blastomere)的研究发现，与芯片技术相比，利用单细胞转录组技术可以多发现数千个基因的表达情况(Nat.Methods 6,377–382,2009)。原本用于单细胞芯片研究的流程被用于单细胞mRNA-Seq测序，但是从单细胞中很难得到较好的数据，不能区分生物本身的差异还是由于由低起始量的RNA造成的技术上的差异；而且mRNA-Seq方法优先扩增mRNA的3’端，不能产生全转录组的覆盖度。In 1990, Norman Iscove's research group first demonstrated the feasibility of transcriptome analysis of single cells. They used PCR technology to achieve exponential amplification of cDNA molecules. In the early 1990s, Eberwine et al. invented a new technology that can obtain cDNA from single living neuronal cells, and then use these cDNAs as templates to transcribe RNA to achieve linear amplification of RNA. With the advent of the chip era, scientists have used these linear and exponential amplification technologies to conduct a large number of comparisons and studies on gene expression differences between single cells. High-throughput RNA sequencing technology emerged in 2008. Soon after, this technology was combined with the previously developed nucleic acid amplification technology to conduct more refined studies of single-cell transcriptomes. In 2009, Tang from the Gurdon Institute of the University of Cambridge in the UK discovered through the study of single mouse cleavage cells (blastomere) that the expression of thousands more genes can be discovered using single-cell transcriptome technology compared with chip technology (Nat. Methods 6, 377–382, 2009). The process originally used for single-cell chip research was used for single-cell mRNA-Seq sequencing, but it is difficult to obtain better data from single cells, and it is impossible to distinguish between differences in the organisms themselves or technology caused by low starting amounts of RNA. differences; and the mRNA-Seq method preferentially amplifies the 3' end of the mRNA and cannot produce full transcriptome coverage.
2012年，Illumina公司联合Sandberg实验室开发出了用于此类分析的领先技术：Smart-seq。这一新方法提高了转录本的覆盖水平，有较高的灵敏度和定量的准确性，增强了可变转录异构体的分析和分辨SNP现象的能力。虽然用单细胞测定估计的表达量有些误差，但是用较少的细胞可以鉴定出来许多不同表达的基因。In 2012, Illumina and Sandberg Laboratory developed a leading technology for such analysis: Smart-seq. This new method improves the coverage level of transcripts, has higher sensitivity and quantitative accuracy, and enhances the ability to analyze variable transcript isoforms and resolve SNP phenomena. Although there is some error in estimating expression levels using single-cell assays, many differentially expressed genes can be identified using fewer cells.
2013年Sandberg实验室于Nature Methods发表Smart-seq2方法(非专利文献1:Simone Picelli,Rickard Sandberg,et al.Smart-seq2for sensitive full-lengthtranscriptome profiling in single cells.nature methods,2013September,doi:10.1038/nmeth.2639.)，此改良技术比Smart-seq产生平均更长的cDNA分子和更高的产量。Smart-seq2转录组文库提高发现能力，增加了覆盖度并且有更低的技术偏差。并且Smart-seq2完全可以采用现有的通用试剂，而非商业化试剂盒，可以很划算地构建测序文库。In 2013, the Sandberg laboratory published the Smart-seq2 method in Nature Methods (Non-patent document 1: Simone Picelli, Rickard Sandberg, et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. nature methods, 2013 September, doi: 10.1038/nmeth .2639.), this improved technology produces on average longer cDNA molecules and higher yields than Smart-seq. Smart-seq2 transcriptome libraries improve discovery capabilities, increase coverage and have lower technical bias. Moreover, Smart-seq2 can completely use existing general-purpose reagents instead of commercial kits, and can construct sequencing libraries very cost-effectively.
Smart-seq2方法使用带有固定序列的oligo-dT、TSO、ISPCR引物进行逆转录扩增，能够有效提高cDNA的扩增效率。但，在采用例如Illumina高通量测序仪进行测序时，从采用该方法构建的高通量测序文库中获取的有效数据的比例偏低，这会给后续的数据分析带来不利影响。The Smart-seq2 method uses oligo-dT, TSO, and ISPCR primers with fixed sequences for reverse transcription amplification, which can effectively improve the amplification efficiency of cDNA. However, when sequencing is performed using an Illumina high-throughput sequencer, for example, the proportion of valid data obtained from the high-throughput sequencing library constructed using this method is low, which will have an adverse impact on subsequent data analysis.
发明内容Contents of the invention
1.一种基于拓扑捕获的RNA样本处理系统，其包括：1. An RNA sample processing system based on topological capture, which includes:
a)作为反应隔室外层的选择性透过膜，所述选择性透过膜能够选择性地透过RNA样本处理试剂； a) As a selectively permeable membrane on the outer layer of the reaction compartment, the selectively permeable membrane can selectively pass through the RNA sample processing reagent;
b)位于所述反应隔室内部的内容物，内容物包括RNA捕获试剂和待分析RNA；其中，b) Contents located inside the reaction compartment, including RNA capture reagents and RNA to be analyzed; wherein,
所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物；The RNA to be analyzed and the RNA capture reagent are connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are transformed through biological or chemical reactions. The nucleic acid content of the formed deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA);
所述选择性透过膜能够选择性地留存RNA捕获试剂和待分析RNA连接而成的整体或复合物或核酸内容物；The selectively permeable membrane can selectively retain the whole or complex or nucleic acid content formed by connecting the RNA capture reagent and the RNA to be analyzed;
所述整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的1/2。The diameter of the whole or complex or nucleic acid content is greater than 1/2 the pore size of the membrane pores of the selectively permeable membrane.
2.根据项1所述的基于拓扑捕获的RNA样本处理系统，其中，2. The RNA sample processing system based on topological capture according to item 1, wherein,
位于所述反应隔室内部的RNA捕获试剂、待分析RNA，以及所述RNA捕获试剂和待分析RNA连接而成的整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物为液态、胶状或半液态；RNA capture reagent, RNA to be analyzed located inside the reaction compartment, and the whole or complex formed by connecting the RNA capture reagent and the RNA to be analyzed, or the deoxyribonucleic acid (DNA) and/or the deoxyribonucleic acid (DNA) formed after the conversion or the nucleic acid content of ribonucleic acid (RNA) is liquid, gelatinous or semi-liquid;
所述反应隔室内部还包含渗透压调节剂；The reaction compartment also contains an osmotic pressure regulator inside;
优选所述渗透压调节剂是葡聚糖。Preferably the osmotic pressure regulator is dextran.
3.根据项1～2中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，3. The RNA sample processing system based on topological capture according to any one of items 1 to 2, wherein,
所述捕获试剂为由带双键的寡核苷酸引物自身聚合而成的胶状高分子化合物或带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物；The capture reagent is a colloidal polymer compound formed by the polymerization of an oligonucleotide primer with a double bond itself or a polymerization of an oligonucleotide primer with a double bond and one or more compound monomers with a double bond. Colloidal polymer compound formed;
所述RNA捕获试剂与所述待分析RNA进行逆转录反应得到直径大于选择性透过膜孔径的1/2的产物分子。The RNA capture reagent performs a reverse transcription reaction with the RNA to be analyzed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
4.根据项3所述的基于拓扑捕获的RNA样本处理系统，其中，4. The RNA sample processing system based on topological capture according to item 3, wherein,
所述RNA捕获试剂与所述待分析RNA进行逆转录反应后，进行PCR扩增反应，得到直径大于选择性透过膜孔径的1/2的产物分子。After performing a reverse transcription reaction between the RNA capture reagent and the RNA to be analyzed, a PCR amplification reaction is performed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
5.根据项1～4中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，5. The RNA sample processing system based on topological capture according to any one of items 1 to 4, wherein,
所述待分析RNA，以及所述RNA捕获试剂和待分析RNA连接成整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物后，进一步地通过以下的第二捕获试剂进行捕获：After the RNA to be analyzed, the RNA capture reagent and the RNA to be analyzed are connected into a whole or a complex, or the nucleic acid content of the deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after the conversion, Further capture is performed with the following second capture reagent:
所述第二捕获试剂为DNA转座酶和DNA的复合物；The second capture reagent is a complex of DNA transposase and DNA;
优选所述DNA转座酶为Tn5，所述DNA在其末端具有转座酶识别序列。Preferably, the DNA transposase is Tn5, and the DNA has a transposase recognition sequence at its end.
6.根据项1～5中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，6. The RNA sample processing system based on topological capture according to any one of items 1 to 5, wherein,
所述待分析RNA来源于同一个细胞或细胞核；优选所述待分析RNA处于完整的 细胞或细胞核中。The RNA to be analyzed originates from the same cell or cell nucleus; preferably, the RNA to be analyzed is in a complete in a cell or nucleus.
7.制造根据项1至6的任一项所述的基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：7. A method for manufacturing the topological capture-based RNA sample processing system according to any one of items 1 to 6, which includes the following steps:
准备包括透压调节剂和第一水性溶剂的第一相；preparing a first phase including a tonicity regulator and a first aqueous solvent;
准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;
RNA捕获试剂添加在第一相或第二相中；RNA capture reagent is added in the first or second phase;
将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；Mix the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；以及Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and
对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；The above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室；Demulsifying the water-in-oil emulsion that has been cured or semi-cured to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the interior;
将外层具有选择性透过膜、在内部具有内容物的反应隔室混合至细胞裂解液以释放细胞或细胞核内的RNA或经过加热释放细胞或细胞核内的RNA从而与RNA捕获试剂接触。A reaction compartment with a selectively permeable membrane on the outside and content on the inside is mixed into a cell lysate to release RNA in cells or nuclei or is heated to release RNA in cells or nuclei to be contacted with an RNA capture reagent.
8.制造基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：8. A method of manufacturing an RNA sample processing system based on topological capture, which includes the following steps:
准备包括RNA捕获试剂、渗透压调节剂和第一水性溶剂的第一相；preparing a first phase including an RNA capture reagent, a tonicity regulator, and a first aqueous solvent;
准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;
将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；Mix the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
将细胞裂解液混合至第一相或第二相；优选细胞裂解液与细胞或细胞核在不同的相；Mix the cell lysate into the first phase or the second phase; preferably the cell lysate and the cells or cell nuclei are in different phases;
将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；且细胞裂解液辅助释放细胞或细胞核内的RNA从而与RNA捕获试剂接触；以及Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and the cell lysis solution assists in releasing RNA in the cells or cell nuclei to thereby Contact with RNA capture reagents; and
对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；The above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室。The water-in-oil emulsion that has been cured or semi-cured is demulsified to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the inside.
9.利用根据项1至6的任一项所述的基于拓扑捕获的RNA样本处理系统制备二代测序(NGS)文库的方法，该方法包括： 9. A method for preparing a next-generation sequencing (NGS) library using the topological capture-based RNA sample processing system according to any one of items 1 to 6, the method comprising:
(a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；(a) Preparing a composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；并且通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于所述第一条cDNA链延伸的条件下，与RNA分子互补，使其与TSO互补；Annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and performing a reverse transcriptase reaction by contacting the RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein The TSO may or may not contain a locked nucleic acid (LNA) at its 3'-end that is complementary to the RNA molecule under conditions suitable for extension of the first cDNA strand, making it complementary to the TSO;
(b)在足以产生产物双链DNA的扩增条件下，对RNA-cDNA中间体进行二链合成扩增，得到第二条DNA；(b) Under amplification conditions sufficient to produce the product double-stranded DNA, perform double-stranded synthetic amplification of the RNA-cDNA intermediate to obtain the second DNA;
(c)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物第二条DNA以产生标记样品；(c) labeling the product with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
(d)将所述加接头DNA片段进行PCR扩增，得到扩增产物；(d) PCR amplify the adapter-added DNA fragment to obtain an amplification product;
RNA样品为项1～6中任一项中涉及的所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物，The RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of items 1 to 6 connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after biological or chemical reaction with the RNA capture reagent,
其中(a)-(d)在项1～6中任一项中涉及的反应隔室中进行。Wherein (a)-(d) are carried out in the reaction compartment involved in any one of items 1 to 6.
10.如项9所述的方法，其中，所述逆转录反应在甲基供体和金属盐存在下进行。10. The method of item 9, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
11.如项11所述的方法，其特征在于，所述甲基供体是甜菜碱。11. The method of item 11, wherein the methyl donor is betaine.
12.如项11所述的方法，其特征在于，所述金属盐是镁盐。12. The method of item 11, wherein the metal salt is a magnesium salt.
13.如项12所述的方法，其中所述镁盐具有7mM以上的浓度。13. The method of item 12, wherein the magnesium salt has a concentration of 7mM or more.
14.如项9所述的方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基以及0个或一个以上锁核酸(LNA)残基。14. The method of item 9, wherein the template switching oligonucleotide comprises one or two ribonucleotide residues and zero or more locked nucleic acid (LNA) residues.
15.如项14所述的方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。15. The method of item 14, wherein the one or two ribonucleotide residues are riboguanine.
16.如项14所述的方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。16. The method of item 14, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
17.如项16所述的方法，其特征在于，所述锁核酸残基是锁鸟嘌呤。17. The method of item 16, wherein the locked nucleic acid residue is locked guanine.
18.如项17所述的方法，其特征在于，所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。18. The method of item 17, wherein the template switching oligonucleotide contains three nucleotide residues characterized by the molecular formula rGrG+N at the 3'-end, where +N represents locked Nucleotide residues.
19.如项18所述的方法，其中所述模板转换寡核苷酸包含rGrG+G。19. The method of item 18, wherein the template switching oligonucleotide comprises rGrG+G.
20.如项10所述的方法，其中所述甲基供体是甜菜碱而所述金属盐是浓度至少为 9mM的MgCl2。20. The method of item 10, wherein the methyl donor is betaine and the metal salt is in a concentration of at least 9mM MgCl2 .
21.如项9所述的方法，其中所述模板转换寡核苷酸选自由以下组成的组：i.rGrG+G，21. The method of item 9, wherein the template switching oligonucleotide is selected from the group consisting of: i.rGrG+G,
优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，Preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N，ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and
iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
22.根据项9所述的方法，其特征在于，所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。22. The method according to item 9, characterized in that the cDNA is in a solution mixed with oligonucleotide primers or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
23.根据项22所述的方法，其中，所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNNNNNN，其中“N”是任何核苷碱基，而“V”是“A”或“C”或“G”。23. The method of item 22, wherein the oligonucleotide primer comprises SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, where "N" is any nucleoside base, and "V" Is it "A" or "C" or "G".
24.一种分析多个单细胞中基因表达的方法，该方法包括以下步骤：根据项9的方法制备cDNA文库；并对cDNA文库进行测序。24. A method for analyzing gene expression in multiple single cells, the method comprising the following steps: preparing a cDNA library according to the method of item 9; and sequencing the cDNA library.
25.一种模板转换寡核苷酸(TSO)，其中其在3'-末端包含一个锁定的核苷酸残基。25. A template switching oligonucleotide (TSO), wherein it contains a locked nucleotide residue at the 3'-end.
26.根据项25所述的模板转换寡核苷酸，其中其在3'-末端包含三个核苷酸残基，所述核苷酸残基选自+N+N+N、N+N+N、NN+N、rN+N+N和rNrN+N，其中每次出现的N独立地是脱氧核糖核苷酸残基，每次出现的rN独立地是核糖核苷酸残基，并且每次出现的+N独立地是锁定的核苷酸残基。26. The template switching oligonucleotide according to item 25, wherein it contains three nucleotide residues at the 3'-end, and the nucleotide residues are selected from +N+N+N, N+N +N, NN+N, rN+N+N, and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue and each occurrence of rN is independently a ribonucleotide residue, and Each occurrence of +N is independently a locked nucleotide residue.
27.根据项25的模板转换寡核苷酸，其中所述锁定核苷酸残基选自锁定的鸟嘌呤、锁定的腺嘌呤、锁定的尿嘧啶、锁定的胸腺嘧啶、锁定的胞嘧啶和锁定的5-甲基胞嘧啶。27. The template switching oligonucleotide according to item 25, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked of 5-methylcytosine.
28.根据项27所述的模板转换寡核苷酸，其中所述三个核苷酸残基选自NN+G、rNrN+G、GG+N、rGrG+G和GG+G。28. The template switching oligonucleotide according to item 27, wherein the three nucleotide residues are selected from the group consisting of NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
29.根据项9所述的方法，其特征在于，所述RNA是从选择性透过膜包围而成的反应隔室中的单细胞中裂解释放出的RNA。29. The method according to item 9, characterized in that the RNA is RNA released from cleaving single cells in a reaction compartment surrounded by a selectively permeable membrane.
30.根据项29所述的单细胞，其特征在于，所述单细胞通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。30. The single cell according to item 29, characterized in that the single cell is wrapped into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
31.根据项9所述的方法，其中所述转座酶为Tn5转座酶。31. The method of item 9, wherein the transposase is Tn5 transposase.
32.根据项31所述的方法，其中所述转座子末端结构域包括Tn5转座子末端结构 域。32. The method of item 31, wherein the transposon end domain comprises a Tn5 transposon end structure area.
33.根据项9所述的方法，其中所述方法进一步包括将所述第一双链产物cDNA与第二双链产物DNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。33. The method of item 9, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product DNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
34.根据项9所述的方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。34. The method of clause 9, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
35.根据项9所述的方法，其中所述的方法都在选择性透过膜包围而成的反应隔室中进行。35. The method according to item 9, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
36.一种在选择性透过膜包围而成的反应隔室中使用核糖核酸(RNA)样品制备二代测序(NGS)文库的方法，该方法包括：36. A method for preparing a next-generation sequencing (NGS) library using ribonucleic acid (RNA) samples in a reaction compartment surrounded by a selectively permeable membrane, the method comprising:
(a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；(a) Preparing a composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；和通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于第一条DNA链延伸的条件下，与RNA分子互补，使其与TSO互补；annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and performing a reverse transcriptase reaction by contacting the RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein The TSO contains or does not contain a locked nucleic acid (LNA) at its 3'-end that is complementary to the RNA molecule under conditions suitable for extension of the first DNA strand, making it complementary to the TSO;
(b)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物mRNA/cDNA杂交双链以产生标记样品；(b) hybridizing double strands with a transposome labeled product mRNA/cDNA comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
(c)将所述加接头DNA片段进行PCR扩增，得到扩增产物，(c) PCR amplify the adapter-added DNA fragment to obtain an amplification product,
RNA样品为项1～5中任一项中涉及的所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物，The RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of items 1 to 5 connected into a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after biological or chemical reaction with the RNA capture reagent,
其中(a)-(c)在项1～6中任一项中涉及的反应隔室中进行。Wherein (a)-(c) are carried out in the reaction compartment involved in any one of items 1 to 6.
37.如项36所述的方法，其中所述逆转录反应在甲基供体和金属盐存在下进行。37. The method of item 36, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
38.如项37所述的方法，其特征在于，所述甲基供体是甜菜碱。38. The method of item 37, wherein the methyl donor is betaine.
39.如项37所述的方法，其特征在于，所述金属盐是镁盐。39. The method of item 37, wherein the metal salt is a magnesium salt.
40.如项37所述的方法，其中所述镁盐具有至少7mM的浓度。40. The method of item 37, wherein the magnesium salt has a concentration of at least 7mM.
41.如项35所述的方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基和所述0个或一个以上锁核酸(LNA)残基。41. The method of item 35, wherein said template switching oligonucleotide comprises one or two ribonucleotide residues and said zero or more locked nucleic acid (LNA) residues.
42.如项41所述的方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。 42. The method of item 41, wherein the one or two ribonucleotide residues are riboguanine.
43.如项41所述的方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。43. The method of item 41, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
44.如项43所述的方法，其特征在于，所述锁核酸残基是锁鸟嘌呤。44. The method of item 43, wherein the locked nucleic acid residue is locked guanine.
45.如项36所述的方法，其特征在于，所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。45. The method of item 36, wherein the template switching oligonucleotide contains three nucleotide residues characterized by the molecular formula rGrG+N at the 3'-end, where +N represents locked Nucleotide residues.
46.如项45所述的方法，其中所述模板转换寡核苷酸包含rGrG+G。46. The method of item 45, wherein the template switching oligonucleotide comprises rGrG+G.
47.根据项37所述的方法，其中所述甲基供体是甜菜碱，而所述金属盐是浓度至少为9mM的MgCl2。47. The method of item 37, wherein the methyl donor is betaine and the metal salt is MgCl2 at a concentration of at least 9mM.
48.如项36所述的方法，其中所述模板转换寡核苷酸选自由以下组成的组：48. The method of item 36, wherein the template switching oligonucleotide is selected from the group consisting of:
i.rGrG+G，优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，i.rGrG+G, preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N，ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and
iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
49.根据项37所述的方法，其特征在于，所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。49. The method according to item 37, characterized in that the cDNA is in a solution mixed with an oligonucleotide primer or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
50.根据项49所述的方法，其中所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNNNNNN，其中“N”是任何核苷碱基，而“V”是“A”或“C”或“G”。50. The method of item 49, wherein the oligonucleotide primer comprises SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N" is any nucleobase and "V" is "A" or "C" or "G".
51.一种分析多个单细胞中基因表达的方法，该方法包括以下步骤：使用根据项36的方法制备cDNA文库；并对cDNA文库进行测序。51. A method for analyzing gene expression in multiple single cells, the method comprising the steps of: preparing a cDNA library using the method according to item 36; and sequencing the cDNA library.
52.一种模板转换寡核苷酸(TSO)，在其3'-最末端包含一个锁定的核苷酸残基。52. A template switching oligonucleotide (TSO) containing a locked nucleotide residue at its 3'-most end.
53.根据项52所述的模板转换寡核苷酸，其在3'-末端包含三个核苷酸残基，所述核苷酸残基选自+N+N+N、N+N+N、NN+N、rN+N+N和rNrN+N，其中每次出现的N独立地是脱氧核糖核苷酸残基，每次出现的rN独立地是核糖核苷酸残基，并且每次出现的+N独立地是锁定的核苷酸残基。53. The template switching oligonucleotide according to item 52, which contains three nucleotide residues at the 3'-end, and the nucleotide residues are selected from +N+N+N, N+N+ N, NN+N, rN+N+N, and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Each occurrence of +N is independently a locked nucleotide residue.
54.项52的模板转换寡核苷酸，其中所述锁定核苷酸残基选自锁定的鸟嘌呤、锁定的腺嘌呤、锁定的尿嘧啶、锁定的胸腺嘧啶、锁定的胞嘧啶和锁定的5-甲基胞嘧啶。54. The template switching oligonucleotide of item 52, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
55.如项53所述的模板转换寡核苷酸，其中所述三个核苷酸残基选自NN+G、 rNrN+G、GG+N、rGrG+G和GG+G。55. The template switching oligonucleotide as described in item 53, wherein the three nucleotide residues are selected from NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
56.如项54所述的方法，其特征在于，所述RNA是选择性透过膜包围而成的反应隔室中的单细胞中裂解释放出的RNA。56. The method of item 54, wherein the RNA is RNA released by cleavage of single cells in a reaction compartment surrounded by a selectively permeable membrane.
57.根据项56所述的单细胞，其特征在于，通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。57. The single cell according to item 56, characterized in that it is packaged into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
58.根据项36所述的方法，其中所述转座酶包括Tn5转座酶。58. The method of item 36, wherein the transposase comprises Tn5 transposase.
59.根据项58所述的方法，其中所述转座子末端结构域包括Tn5转座子末端结构域。59. The method of item 58, wherein the transposon terminus domain comprises a Tn5 transposon terminus domain.
60.根据项36所述的方法，其中所述方法进一步包括将第一双链产物cDNA与第二双链产物cDNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。60. The method of item 36, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product cDNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
61.根据项36所述的方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。61. The method of clause 36, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
62.根据项36所述的方法，其中所述方法都在选择性透过膜包围而成的反应隔室中进行。62. The method according to item 36, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
本申请的技术方案取得了下述有益效果：The technical solution of this application has achieved the following beneficial effects:
1.通过“半透性隔室”实现纯化、清洗或多步反应所需要的各种反应物质交换；2.通过捕获试剂富集特异性的生物分子；3.进而实现高通量的单细胞全长转录组测序。1. Through the "semi-permeable compartment", exchange of various reaction substances required for purification, cleaning or multi-step reactions; 2. Enrichment of specific biomolecules through capture reagents; 3. To achieve high-throughput single cells Full-length transcriptome sequencing.
附图说明Description of the drawings
图1A.利用微流控系统形成包裹带双键的寡核苷酸(引物)与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物和细胞的选择性透过膜包围而成的反应隔室。Figure 1A. Microfluidic system is used to form a colloidal polymer composed of an oligonucleotide (primer) with a double bond and one or more compound monomers with a double bond and the selective permeation of cells. A reaction compartment surrounded by a membrane.
图1B.利用微流控系统形成包裹细胞的选择性透过膜包围而成的反应隔室。Figure 1B. A microfluidic system is used to form a reaction compartment surrounded by a selectively permeable membrane surrounding cells.
图2A.所述待分析物为DNA分子，所述捕获试剂可为蛋白质、核酸中的一种或两种以上以及它们共同形成的复合物。Figure 2A. The analyte is a DNA molecule, and the capture reagent can be one or more of proteins, nucleic acids, and complexes formed by them together.
图2B.所述待分析物为RNA/DNA杂交链分子，所述捕获试剂可为蛋白质、核酸中的一种或两种以上以及它们共同形成的复合物。Figure 2B. The analyte is an RNA/DNA hybrid chain molecule, and the capture reagent can be one or more of proteins, nucleic acids, and complexes formed by them together.
图2C.所述待分析物为RNA分子，所述捕获试剂可为由带双键的寡核苷酸(引物)与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物。Figure 2C. The analyte is an RNA molecule, and the capture reagent can be a colloidal polymer polymerized by an oligonucleotide (primer) with a double bond and one or more compound monomers with a double bond. Molecular compounds.
图2D.所述待分析物为RNA分子，所述捕获试剂可为寡核苷酸(引物)。Figure 2D. The analyte is an RNA molecule, and the capture reagent can be an oligonucleotide (primer).
图3.产生含有双水相及靶向捕获试剂的液滴的微流体装置系统。 Figure 3. Microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
图4.通过微流体装置系统向微流控芯片中注入第一种流体(I相溶液，富含葡聚糖)、第二种流体(II相溶液，富含基于聚乙二醇的聚合物)、连续相(载体油为氟化油并且包含表面活性剂，例如PFPE-PEG-PFPE(全氟聚醚-聚乙二醇-全氟聚醚)三嵌段共聚物)；靶向捕获试剂从第一种流体、第二种流体、连续相(载体油)或任意两者或三者进入微流控体系。Figure 4. The first fluid (phase I solution, rich in dextran) and the second fluid (phase II solution, rich in polyethylene glycol-based polymer) are injected into the microfluidic chip through the microfluidic device system. ), continuous phase (the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer); target capture reagent Enter the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.
图5.通过引发聚合来硬化外层II相的方法。Figure 5. Method of hardening outer phase II by initiating polymerization.
图6.破乳释放由选择性透过膜包围而成的反应隔室和样本制备系统。Figure 6. Demulsification release reaction compartment and sample preparation system surrounded by selectively permeable membrane.
图7.选择性透过膜包围而成的反应隔室。Figure 7. Reaction compartment surrounded by selectively permeable membrane.
图8.成胶后的选择性透过膜包围而成的反应隔室。Figure 8. The reaction compartment surrounded by the selectively permeable membrane after gel formation.
图9.破乳后的选择性透过膜包围而成的反应隔室。Figure 9. Reaction compartment surrounded by selectively permeable membrane after demulsification.
图10.反应隔室中的mRNA捕获微球和细胞；微球～2.7微米；外壁孔径约50nm。Figure 10. mRNA capture microspheres and cells in the reaction compartment; microspheres ~2.7 microns; outer wall pore size ~50nm.
图11.选择性透过膜包围而成的反应隔室内的电镜照片。Figure 11. Electron microscope photo of the reaction compartment surrounded by a selectively permeable membrane.
图12.选择性透过膜包围而成的反应隔室内进行细胞裂解。Figure 12. Cell lysis occurs in a reaction compartment surrounded by a selectively permeable membrane.
图13.选择性透过膜包围而成的反应隔室内进行去除基因组DNA。Figure 13. Removal of genomic DNA occurs in a reaction compartment surrounded by a selective permeable membrane.
图14.选择性透过膜包围而成的反应隔室内进行单细胞mRNA反转录cDNA扩增的荧光染色。Figure 14. Fluorescent staining of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
图15.选择性透过膜包围而成的反应隔室内单细胞mRNA反转录cDNA扩增的碱溶解的琼脂糖凝胶。Figure 15. Alkali-dissolved agarose gel for single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
图16.选择性透过膜包围而成的反应隔室内单细胞mRNA反转录cDNA扩增的Qsep表征。Figure 16. Qsep characterization of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment surrounded by a selectively permeable membrane.
图17.选择性透过膜包围而成的反应隔室内进行单细胞mRNA反转录cDNA扩增，进行namocell分选。Figure 17. Single-cell mRNA reverse transcription cDNA amplification and namocell sorting are performed in a reaction compartment surrounded by a selectively permeable membrane.
图18.选择性透过膜包围而成的反应隔室内的cDNA扩增文库打断。Figure 18. cDNA amplification library fragmentation in a reaction compartment surrounded by a selectively permeable membrane.
图19.选择性透过膜包围而成的反应隔室内的cDNA扩增文库打断的Qsep表征。Figure 19. Qsep characterization of cDNA amplification library interruption in a reaction compartment surrounded by a selectively permeable membrane.
图20.选择性透过膜包围而成的反应隔室内的RNA/DNA杂交链打断。Figure 20. Interruption of RNA/DNA hybrid strands in a reaction compartment surrounded by a selectively permeable membrane.
图21.选择性透过膜包围而成的反应隔室内的RNA/DNA杂交链打断的Bioanalzyer 2100表征。Figure 21. Bioanalzyer 2100 characterization of RNA/DNA hybrid chain disruption in a reaction compartment surrounded by a selectively permeable membrane.
图22.在选择性透过膜组成的反应隔室成胶前(磁珠拓扑捕获)。Figure 22. Before gelation of a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
图23.在选择性透过膜组成的反应隔室成胶后(磁珠拓扑捕获)。Figure 23. After gelation of the reaction compartment consisting of a selectively permeable membrane (magnetic bead topological capture).
图24.在选择性透过膜组成的反应隔室破乳后(磁珠拓扑捕获)。 Figure 24. After demulsification of reaction compartments composed of selectively permeable membranes (magnetic bead topological capture).
图25.在选择性透过膜组成的反应隔室内进行细胞裂解(磁珠拓扑捕获)。Figure 25. Cell lysis in a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
图26.在选择性透过膜组成的反应隔室内内进行去除基因组DNA(磁珠拓扑捕获)。Figure 26. Removal of genomic DNA (magnetic bead topological capture) in a reaction compartment composed of a selectively permeable membrane.
图27.在选择性透过膜组成的反应隔室内进行单细胞mRNA反转录cDNA扩增的荧光染色(磁珠拓扑捕获)。Figure 27. Fluorescent staining of single-cell mRNA reverse-transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane (magnetic bead topological capture).
具体实施方式Detailed ways
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a thorough understanding of the invention, and to fully convey the scope of the invention to those skilled in the art.
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。It should be noted that certain words are used in the description and claims to refer to specific components. Those skilled in the art will understand that skilled persons may use different names to refer to the same component. This specification and the claims do not use difference in nouns as a way to distinguish components, but rather use differences in functions of the components as a criterion for distinction. If the words "include" or "include" mentioned throughout the description and claims are open-ended terms, they should be interpreted as "include but not limited to." The following descriptions of the description are preferred embodiments for implementing the present invention. However, the descriptions are for the purpose of general principles of the description and are not intended to limit the scope of the present invention. The protection scope of the present invention shall be determined by the appended claims.
如本文所用，就特定组分而言“基本上不含”在本文中用于表示特定组分未被有目的地配制到组合物中和/或仅作为污染物或以痕量存在。因此，由组合物的任何意外污染导致的特定组分的总量低于0.05％，优选低于0.01％。最优选的是其中特定组分的量用标准分析方法检测不到的组合物。As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the specific component is present in an amount undetectable by standard analytical methods.
如在本说明书中所使用的，“一”或“一个”可以表示一个或多个。如权利要求中所使用的，当与单词“包含”一起使用时，单词“一”或“一个”可以表示一个或多于一个。As used in this specification, "a" or "an" may mean one or more. As used in the claims, the word "a" or "an" when used with the word "comprising" can mean one or more than one.
在权利要求中使用术语“或”用于表示“和/或”，除非明确指出仅指代替代方案或者替代方案是相互排斥的，尽管本申请的内容支持仅指代替代方案和“和/或”的定义。如本文所用，“另一个”可以表示至少第二个或更多个。The term "or" is used in the claims to mean "and/or" unless it is expressly stated that only the alternatives are to be referred to or that the alternatives are mutually exclusive, although the content of this application supports that only the alternatives and "and/or" are to be referred to. "Definition. As used herein, "another" may mean at least a second or more.
贯穿本申请，术语“约”用于指示值包括装置的误差的固有变化，该方法用于测定该值或存在于研究对象之间的变化。Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error of the device, the method used to determine the value, or the variation that exists between study subjects.
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到 具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。The ways to obtain various biological materials described in the examples are only to provide an experimental way to achieve The specific purpose of disclosure should not be a limitation on the source of the biological material of the present invention. In fact, the sources of biological materials used are wide, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the embodiments.
本申请在第一方面提供了一种基于拓扑捕获的RNA样本处理系统。In a first aspect, the present application provides an RNA sample processing system based on topological capture.
在一个具体实施方式中，提供了一种基于拓扑捕获的RNA样本处理系统，其包括：In a specific embodiment, a topological capture-based RNA sample processing system is provided, which includes:
a)作为反应隔室外层的选择性透过膜，所述选择性透过膜能够选择性地透过RNA样本处理试剂；b)位于所述反应隔室内部的内容物，内容物包括RNA捕获试剂和待分析RNA；其中，所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物；所述选择性透过膜能够选择性地留存RNA捕获试剂和待分析RNA连接而成的整体或复合物或核酸内容物；所述整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的1/2。有利地，所得到的整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的0.5倍、0.6倍、0.7倍、3/4、0.8倍、0.9倍、1倍、1.5倍、2倍、2.5倍、3倍或更大。a) a selectively permeable membrane as the outer layer of the reaction compartment, the selectively permeable membrane capable of selectively transmitting RNA sample processing reagents; b) the content located inside the reaction compartment, the content including RNA capture Reagent and RNA to be analyzed; wherein, the RNA to be analyzed and the RNA capture reagent are connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are The nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after conversion is generated through biological or chemical reactions; the selectively permeable membrane can selectively retain the RNA capture reagent and connect with the RNA to be analyzed. The whole body or complex or nucleic acid content; the diameter of the whole body or complex or nucleic acid content is greater than 1/2 of the pore diameter of the membrane pores of the selectively permeable membrane. Advantageously, the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
在本申请的上下文中，“反应隔室”意指一种半封闭的反应空间，其由半透膜包围而成，可以进行部分物质(根据其分子大小的不同、疏水性/亲水性的不同，携带电荷的不同而不同等)的交换，特别地针对本申请，相对于半透膜的孔径较小的分子可以自由进入或退出所述反应隔室，相对于半透膜的孔径较大的分子则可以被截留在反应隔室内。在本申请的上下文中，“选择性透过膜”有时也写作“半透膜”，特指可以在该膜两侧根据物质的分子大小的不同、疏水性/亲水性的不同，携带电荷的不同等物理/化学因素选择性地允许物质通过的膜结构。在本说明书的上下文中，“半透膜膜孔的孔径”可以理解为在半透膜的各个膜孔上距离最远的两个点的实际距离的平均值。在本说明书的上下文中，如无特别说明，核酸特指脱氧核糖核酸(DNA)和核糖核酸(RNA)。在本说明书的上下文中，“直径”的对应英文是equivalent diameter，更具体地，可以理解为“斯托克斯直径”，意指当所研究的颗粒物与某一球状体具有相同的最终沉降速度时，该球状体的直径即为所研究颗粒物的斯托克斯直径。In the context of this application, "reaction compartment" means a semi-enclosed reaction space surrounded by a semi-permeable membrane that allows the reaction of some substances (depending on their molecular size, hydrophobicity/hydrophilicity) Different, carry different charges, etc.) exchange, specifically for this application, molecules with a smaller pore size relative to the semipermeable membrane can freely enter or exit the reaction compartment, relative to the larger pore size of the semipermeable membrane molecules can be trapped within the reaction compartment. In the context of this application, "selective permeable membrane" is sometimes also written as "semi-permeable membrane", specifically referring to the ability to carry charges on both sides of the membrane based on the difference in molecular size and hydrophobicity/hydrophilicity of the substance. A membrane structure that selectively allows substances to pass through due to different physical/chemical factors. In the context of this specification, "the pore size of a semipermeable membrane pore" may be understood as the average of the actual distances between the two furthest points on each membrane pore of the semipermeable membrane. In the context of this specification, unless otherwise stated, nucleic acid specifically refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In the context of this specification, the English equivalent of "diameter" is equivalent diameter, more specifically, it can be understood as "Stokes diameter", which means when the particle under study has the same final settling velocity as a certain spheroidal body , the diameter of the spheroids is the Stokes diameter of the particles under study.
在又一具体实施方式中，提供了上述基于拓扑捕获的RNA样本处理系统，其中，位于所述反应隔室内部的RNA捕获试剂、待分析RNA，以及所述RNA捕获试剂和待分析RNA连接而成的整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核 糖核酸(RNA)的核酸内容物为液态、胶状或半液态；所述反应隔室内部还包含渗透压调节剂；优选所述渗透压调节剂是葡聚糖。In yet another specific embodiment, the above-mentioned RNA sample processing system based on topological capture is provided, wherein the RNA capture reagent and the RNA to be analyzed are located inside the reaction compartment, and the RNA capture reagent and the RNA to be analyzed are connected and The whole or complex formed, or the deoxyribonucleic acid (DNA) and/or nuclei formed after said transformation The nucleic acid content of RNA is in a liquid, colloidal or semi-liquid state; the inside of the reaction compartment also contains an osmotic pressure regulator; preferably, the osmotic pressure regulator is dextran.
在本说明书的上下文中，“拓扑捕获”是本领域专业术语，“拓扑”对应英文topology。拓扑捕获是超分子化学和拓扑学结合的新概念：基于非共价键作用的机械互锁结构，由选择透过性膜与颗粒组合构成，用于靶向留存生物分子。具体到本申请，即形成一个呈“网兜状”的半透膜结构，其对于直径大的分子(直径大一般对应于分子量大的分子，但与分子的形状也有关)具有明确的截留作用，该截留作用可以通过“网兜状半透膜”的膜孔的孔径进行调整。在再一实施方式中，提供了上述基于拓扑捕获的RNA样本处理系统，其中，所述捕获试剂为由带双键的寡核苷酸引物自身聚合而成的胶状高分子化合物或带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物；In the context of this specification, "topology capture" is a professional term in this field, and "topology" corresponds to the English topology. Topological trapping is a new concept combining supramolecular chemistry and topology: a mechanical interlocking structure based on non-covalent bonding, composed of a combination of selectively permeable membranes and particles, used to target and retain biomolecules. Specific to this application, a "mesh-shaped" semi-permeable membrane structure is formed, which has a clear interception effect on molecules with large diameters (large diameters generally correspond to molecules with large molecular weight, but it is also related to the shape of the molecules). This interception can be adjusted by the pore size of the "mesh-shaped semipermeable membrane". In yet another embodiment, the above-mentioned RNA sample processing system based on topological capture is provided, wherein the capture reagent is a colloidal polymer compound formed by self-polymerization of an oligonucleotide primer with a double bond or a polymer with a double bond. A colloidal polymer compound formed by polymerizing an oligonucleotide primer with one or more compound monomers with double bonds;
所述RNA捕获试剂与所述待分析RNA进行逆转录反应得到直径大于选择性透过膜孔径的1/2的产物分子。有利地，所得到的整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的0.5倍、0.6倍、0.7倍、3/4、0.8倍、0.9倍、1倍、1.5倍、2倍、2.5倍、3倍或更大。The RNA capture reagent performs a reverse transcription reaction with the RNA to be analyzed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane. Advantageously, the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
在本说明书的上下文中，“带双键的寡核苷酸引物”特指一种“双重作用引物”，其既具有一般PCR引物作用，也具有“通过缩聚形成更大直径的分子，进而用作针对待分析RNA的捕获试剂”作用。在又一具体实施方式中，提供了上述基于拓扑捕获的RNA样本处理系统，其中，In the context of this specification, "oligonucleotide primer with double bonds" specifically refers to a "dual-action primer" that not only has the function of a general PCR primer, but also has the function of "forming larger-diameter molecules through condensation, and then using Acts as a capture reagent for the RNA to be analyzed. In yet another specific embodiment, the above-mentioned topological capture-based RNA sample processing system is provided, wherein,
所述RNA捕获试剂与所述待分析RNA进行逆转录反应后，进行PCR扩增反应，得到直径大于选择性透过膜孔径的1/2的产物分子。有利地，所得到的整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的0.5倍、0.6倍、0.7倍、3/4、0.8倍、0.9倍、1倍、1.5倍、2倍、2.5倍、3倍或更大。After performing a reverse transcription reaction between the RNA capture reagent and the RNA to be analyzed, a PCR amplification reaction is performed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane. Advantageously, the diameter of the resulting whole body or complex or nucleic acid content is greater than 0.5 times, 0.6 times, 0.7 times, 3/4, 0.8 times, 0.9 times, 1 times the pore diameter of the membrane pores of the selectively permeable membrane. times, 1.5 times, 2 times, 2.5 times, 3 times or greater.
在说明书的上下文中，“待分析RNA”特指mRNA，其在逆转录酶和适当溶媒、温度的作用下可以发生逆转录反应生成cDNA，进而形成mRNA/cDNA杂交链。所述逆转录反应可以在逆转录PCR仪器中进行。在本说明书的上下文中，PCR是“聚合酶链式反应”的英文缩写。PCR是利用DNA在体外摄氏95℃高温时变性会变成单链，低温(经常是60℃左右)时引物与单链按碱基互补配对的原则结合，再调温度至DNA聚合酶最适反应温度(72℃左右)，DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。基于聚合酶制造的PCR仪实际就是一个温控设备，能在变性温度，复性温度，延伸温 度之间精准地进行控制。通过DNA聚合酶和温度循环的共同作用完成对作为底物的微量核酸的翻倍复制扩增。In the context of the instructions, "RNA to be analyzed" specifically refers to mRNA, which can undergo a reverse transcription reaction under the action of reverse transcriptase, appropriate solvent, and temperature to generate cDNA, thereby forming an mRNA/cDNA hybrid chain. The reverse transcription reaction can be performed in a reverse transcription PCR instrument. In the context of this specification, PCR is the English abbreviation of "polymerase chain reaction". PCR uses DNA to denature into a single strand at a high temperature of 95°C in vitro. At low temperature (usually around 60°C), the primers are combined with the single strand according to the principle of base pairing, and then the temperature is adjusted to the optimal reaction of the DNA polymerase. temperature (around 72°C), DNA polymerase synthesizes complementary strands along the direction from phosphate to five-carbon sugar (5'-3'). The PCR machine based on polymerase is actually a temperature control device, which can adjust the temperature at denaturation temperature, renaturation temperature, and extension temperature. Control accurately between degrees. Through the combined action of DNA polymerase and temperature cycling, the doubling, replication and amplification of trace amounts of nucleic acids as substrates are completed.
在一个具体实施方式中，提供了上述基于拓扑捕获的RNA样本处理系统，其中，In a specific embodiment, the above-mentioned topological capture-based RNA sample processing system is provided, wherein,
所述待分析RNA，以及所述RNA捕获试剂和待分析RNA连接成整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物后，进一步地通过以下的第二捕获试剂进行捕获：所述第二捕获试剂为DNA转座酶和DNA的复合物；优选所述DNA转座酶为Tn5，所述DNA在其末端具有转座酶识别序列，特别地是在两端带有19bp的ME序列的转座酶。在此，Tn5转座酶可以作为第二捕获试剂(形成更大直径的待截留分子)的机制是Tn5的3’-OH亲核攻击靶序列，在转座子插入位点之间形成9bp的粘性末端，转座子的3’-OH同靶DNA的5’-P之间形成共价键。Tn5基于其二聚体蛋白互作的亲和力与打断后的短片段DNA形成复合物，保持原DNA分子的顺序和邻近性。)After the RNA to be analyzed, the RNA capture reagent and the RNA to be analyzed are connected into a whole or a complex, or the nucleic acid content of the deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after the conversion, Capture is further performed by the following second capture reagent: the second capture reagent is a complex of DNA transposase and DNA; preferably, the DNA transposase is Tn5, and the DNA has transposase recognition at its end. sequence, specifically a transposase with a 19 bp ME sequence at both ends. Here, the mechanism by which Tn5 transposase can serve as a second capture reagent (to form a larger diameter molecule to be trapped) is that the 3'-OH of Tn5 nucleophilically attacks the target sequence, forming a 9 bp gap between the transposon insertion sites. At the sticky end, a covalent bond is formed between the 3'-OH of the transposon and the 5'-P of the target DNA. Based on the affinity of its dimer protein interaction, Tn5 forms a complex with the fragmented short DNA fragments, maintaining the order and contiguity of the original DNA molecules. )
在一个具体实施方式中，提供了一种基于拓扑捕获的RNA样本处理系统，其中，所述待分析RNA来源于同一个细胞或细胞核；优选所述待分析RNA处于完整的细胞或细胞核中。在本说明书的上下文中，所述待分析RNA先处于完整的细胞或细胞核中，而后经历细胞裂解的阶段，在被捕获试剂捕获时，处于从细胞/细胞核中释放出的状态。In a specific embodiment, an RNA sample processing system based on topological capture is provided, wherein the RNA to be analyzed originates from the same cell or nucleus; preferably, the RNA to be analyzed is in a complete cell or nucleus. In the context of this specification, the RNA to be analyzed is first in intact cells or nuclei, then undergoes a cell lysis stage, and is in a state of being released from the cells/nuclei when captured by the capture reagent.
本申请在第二方面涉及一种制造上述基于拓扑捕获的RNA样本处理系统的方法。In a second aspect, the present application relates to a method of manufacturing the above-mentioned topological capture-based RNA sample processing system.
在一个具体实施方式中，提供了制造上述基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：准备包括透压调节剂和第一水性溶剂的第一相；准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；In a specific embodiment, a method for manufacturing the above-mentioned topological capture-based RNA sample processing system is provided, which includes the following steps: preparing a first phase including a tonicity regulator and a first aqueous solvent; preparing a mixture of selective permeability a second phase of film-forming material and a second aqueous solvent;
RNA捕获试剂添加在第一相或第二相中；将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；以及对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；The RNA capture reagent is added to the first phase or the second phase; the RNA to be analyzed in intact cells or cell nuclei is mixed into the first phase or the second phase; preferably the RNA to be analyzed in intact cells or cell nuclei is mixed to the first phase; mix the first phase and the second phase into a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and conduct the above-mentioned water-in-oil emulsion. The curing or semi-curing reaction forms a selectively permeable membrane;
对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室；将外层具有选择性透过膜、在内部具有内容物的反应隔室混合至细胞裂解液以释放细胞或细胞核内的RNA或经过加热释放细胞或细胞核内的RNA从而与RNA捕获试剂接触。Demulsify the water-in-oil emulsion that has been cured or semi-cured to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content inside; The reaction compartment with its contents inside is mixed into a cell lysis solution to release RNA within the cells or nuclei or is heated to release RNA within the cells or nuclei into contact with the RNA capture reagent.
所述第一水性溶剂包含下组中的一项或多项：乙醇、甲醛、聚乙烯醇、葡聚糖、羟丙基淀粉、Ficoll、甲氧基聚乙二醇、聚乙二醇、右旋糖酐、磷酸钾、葡萄糖、其他无 机盐(K+,Na+,Li+,(NH4)+,PO4 3–,SO4 2-)、聚乙二醇、聚丙二醇、乙基羟乙基纤维素、环氧乙烷-环氧丙烷、聚(N-异丙基丙烯酰胺)、聚(甲基丙烯酸甲酯-co-甲基丙烯酸)；The first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , potassium phosphate, glucose, other none Organic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl cellulose, ethylene oxide- Propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);
或者or
所述第二水性溶剂包含下组中的一项或多项：乙醇、甲醛、聚乙烯醇、葡聚糖、羟丙基淀粉、Ficoll、甲氧基聚乙二醇、聚乙二醇、右旋糖酐、磷酸钾、葡萄糖、其他无机盐(K+,Na+,Li+,(NH4)+,PO4 3–,SO4 2-)、聚乙二醇、聚丙二醇、乙基羟乙基纤维素、环氧乙烷-环氧丙烷、聚(N-异丙基丙烯酰胺)、聚(甲基丙烯酸甲酯-co-甲基丙烯酸)；The second aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber Element, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);
优选所述第一水性溶剂与第二水性溶剂均为水；Preferably, both the first aqueous solvent and the second aqueous solvent are water;
进一步优选所述渗透压调节剂是葡聚糖且所述葡聚糖在第一水性溶剂中的浓度为3％-10％；最优选所述渗透压调节剂是葡聚糖且所述葡聚糖在第一水性溶剂中的浓度为5.5％；It is further preferred that the osmotic pressure regulator is dextran and the concentration of dextran in the first aqueous solvent is 3%-10%; most preferably the osmotic pressure regulator is dextran and the dextran The concentration of sugar in the first aqueous solvent is 5.5%;
进一步优选所述油相溶剂选自下组中的一种或多种：全氟聚醚-聚乙二醇-全氟聚醚三嵌段共聚物、HFE-7500氟化油、Squalane油、硅油和矿物油；It is further preferred that the oil phase solvent is selected from one or more of the following groups: perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer, HFE-7500 fluorinated oil, Squalane oil, silicone oil and mineral oil;
最优选所述油相溶剂为全氟聚醚-聚乙二醇-全氟聚醚三嵌段共聚物。Most preferably, the oil phase solvent is a perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer.
所述固化或半固化反应的调节条件为紫外光的光照强度和光照时间；The adjustment conditions for the curing or semi-curing reaction are the illumination intensity and illumination time of ultraviolet light;
优选所述固化或半固化反应所使用的催化剂为选自下组的一个或多个的TEMED引发剂：2-羟基-2-甲基-1-苯基丙酮、1-羟基环己基苯基甲酮、2-甲基-2-(4-吗啉基)-1-[4-(甲硫基)苯基]-1-丙酮、2,4,6-三甲基苯甲酰基-二苯基氧化膦、2,4,6-三甲基苯甲酰基苯基膦酸乙酯、2-二甲氨基-2-苄基-1-[4-(4-吗啉基)苯基]-1-丁酮、2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮、MBF苯甲酰甲酸甲酯、苯偶姻及衍生物(安息香、安息香双甲醚、安息香乙醚、安息香异丙醚、安息香丁醚)、苯偶酰类(二苯基乙酮、α，α-二甲氧基-α-苯基苯乙酮)、烷基苯酮类(α，α-二乙氧基苯乙酮、α-羟烷基苯酮、α-胺烷基苯酮)、酰基磷氧化物(芳酰基膦氧化物、双苯甲酰基苯基氧化膦)、二苯甲酮类(二苯甲酮、2,4-二羟基二苯甲酮、米蚩酮)、硫杂蒽酮类(硫代丙氧基硫杂蒽酮、异丙基硫杂蒽酮)；二芳基碘鎓盐、三芳基碘鎓盐、烷基碘鎓盐、异丙苯茂铁六氟磷酸盐；Preferably, the catalyst used in the curing or semi-curing reaction is one or more TEMED initiators selected from the following group: 2-hydroxy-2-methyl-1-phenyl acetone, 1-hydroxycyclohexylphenylmethyl Ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2,4,6-trimethylbenzoyl-diphenyl Phosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl]- 1-Butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, MBF methyl benzoylformate, benzoin and derivatives ( Benzoin, benzoin dimethyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin butyl ether), benzoyl (diphenyl ethyl ketone, α, α-dimethoxy-α-phenylacetophenone), alkanes Benzophenones (α, α-diethoxyacetophenone, α-hydroxyalkylphenone, α-aminoalkylphenone), acylphosphorus oxide (aroylphosphine oxide, bisbenzoylbenzene phosphine oxide), benzophenones (benzophenone, 2,4-dihydroxybenzophenone, Michinone), thioxanthone (thiopropoxythioxanthone, isopropyl Thiaxanthone); diaryliodonium salt, triaryliodonium salt, alkyl iodonium salt, cumene ferrocene hexafluorophosphate;
所述选择性透过膜形成材料选自下组中的一个或多个：聚烯烃、烯烃共聚物、丙烯酸类、乙烯基聚合物、聚酯、聚碳酸酯、聚酰胺、聚酰亚胺、甲醛树脂、聚氨酯、醚聚合物、纤维素、热塑性弹性体和热塑性聚氨酯材料；The selectively permeable membrane forming material is selected from one or more of the following group: polyolefin, olefin copolymer, acrylic, vinyl polymer, polyester, polycarbonate, polyamide, polyimide, Formaldehyde resin, polyurethane, ether polymer, cellulose, thermoplastic elastomer and thermoplastic polyurethane materials;
优选选择性透过膜形成材料为水凝胶骨架材料；进一步优选所述选择性透过膜形成材料为聚乙二醇二丙烯酸酯(PEGDA)，进一步优选所述PEGDA在第二水性溶剂中的浓 度为1％-10％，Preferably, the selectively permeable membrane-forming material is a hydrogel framework material; further preferably, the selectively permeable membrane-forming material is polyethylene glycol diacrylate (PEGDA), and further preferably, the PEGDA is in a second aqueous solvent. concentrated Degree is 1%-10%,
最优选其为PEGDA且所述PEGDA在第二水性溶剂中的浓度为3％。Most preferably it is PEGDA and the concentration of PEGDA in the second aqueous solvent is 3%.
在本文的上下文中，破乳可以通过使用表面活性剂震荡混匀随后高速离心分离实现。In the context of this article, demulsification can be achieved by shaking the mixture with surfactants followed by high-speed centrifugation.
在又一具体实施方式中，提供了制造上述基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：准备包括RNA捕获试剂、渗透压调节剂和第一水性溶剂的第一相；准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；In yet another specific embodiment, a method for manufacturing the above topological capture-based RNA sample processing system is provided, which includes the following steps: preparing a first phase including an RNA capture reagent, an osmotic pressure regulator and a first aqueous solvent; preparing to mix A second phase of selectively permeable membrane-forming material and a second aqueous solvent; mixing the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mixing the RNA in intact cells or cell nuclei The RNA to be analyzed is mixed into the first phase;
将细胞裂解液混合至第一相或第二相；优选细胞裂解液与细胞或细胞核在不同的相；将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；且细胞裂解液辅助释放细胞或细胞核内的RNA从而与RNA捕获试剂接触；以及对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室。Mix the cell lysate into the first phase or the second phase; preferably the cell lysate and the cells or cell nuclei are in different phases; mix the first phase and the second phase into a mixed hydrophilic phase, and then mix the mixed hydrophilic phase Mix with the oily solvent to prepare a water-in-oil emulsion; and the cell lysate assists in releasing the RNA in the cells or nuclei to contact the RNA capture reagent; and the above-mentioned water-in-oil emulsion is cured or semi-cured to form a selectively permeable emulsion. Passing the membrane; demulsifying the water-in-oil emulsion that has been cured or semi-cured to obtain a reaction compartment with a selectively permeable membrane on the outer layer and content inside.
本申请在第三方面涉及基于拓扑捕获的RNA样本处理系统制备二代测序(NGS)文库的方法。In a third aspect, the present application relates to a method for preparing a next-generation sequencing (NGS) library based on a topological capture-based RNA sample processing system.
在一个具体实施方式中提供了一种基于拓扑捕获的RNA样本处理系统制备二代测序(NGS)文库的方法，该方法包括：(a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；并且通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于所述第一条cDNA链延伸的条件下，与RNA分子互补，使其与TSO互补；(b)在足以产生产物双链DNA的扩增条件下，对RNA-cDNA中间体进行二链合成扩增，得到第二条DNA；(c)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物第二条DNA以产生标记样品；In a specific embodiment, a method for preparing a next-generation sequencing (NGS) library based on an RNA sample processing system based on topological capture is provided. The method includes: (a) preparing a composition including: RNA sample; first strand complementary deoxyribonucleic acid (cDNA) primers; template switch oligonucleotides; reverse transcriptase; and dNTPs; annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and by making the RNA - The cDNA intermediate is contacted with a template switching oligonucleotide (TSO) to perform the reverse transcriptase reaction, wherein the TSO contains or does not contain a locking nucleic acid (LNA) at its 3'-end, in a position suitable for the first cDNA strand Under extension conditions, it is complementary to the RNA molecule, making it complementary to TSO; (b) Under amplification conditions sufficient to produce the product double-stranded DNA, perform two-strand synthesis amplification of the RNA-cDNA intermediate to obtain the second DNA ; (c) labeling the product with a second piece of DNA using a transposome comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
(d)将所述加接头DNA片段进行PCR扩增，得到扩增产物；(d) PCR amplify the adapter-added DNA fragment to obtain an amplification product;
RNA样品为上文所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA) 的核酸内容物，The RNA sample is the above-mentioned RNA to be analyzed and the RNA capture reagent connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are biologically or A chemical reaction that produces deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) that is transformed The nucleic acid content of
其中(a)-(d)在上文所述的反应隔室中进行。wherein (a)-(d) are carried out in the reaction compartment described above.
在本说明书的上下文中，二代测序的称谓是为了与20世纪70年代问世的第一代DNA测序技术相区别，因此也称为“下一代测序”；由于可以同时对数百万乃至数百亿条不同的DNA片段进行测序，该技术也称作“高通量测序”(本申请中的核酸“打断”步骤即是为高通量测序做准备)。二代测序使DNA测序的成本下降了至少5个数量级，使得它被广泛应用在生物学的各个领域。根据测序原理，二代测序通常分为边合成边测序和边连接边测序两类。本申请的二代测序是基于边合成边测序。在本说明书的上下文中，LNA是locked nucleic acid的英文缩写，中文称“锁核酸”或“锁定核酸”。LNA可以与DNA或RNA结合形成杂合寡核苷酸分子，因此通过LNA引入量可以改善PCR反应、微阵列芯片以及原位杂交等多种基于杂交原理的技术。这类核酸可以增加引物或探针的解链问题，加强这些物质的稳定性，可应用在实时荧光定量PCR等技术中。In the context of this specification, the name of second-generation sequencing is to distinguish it from the first-generation DNA sequencing technology that came out in the 1970s, so it is also called "next-generation sequencing"; because it can simultaneously analyze millions or even hundreds of Billions of different DNA fragments are sequenced, and this technology is also called "high-throughput sequencing" (the nucleic acid "interruption" step in this application is to prepare for high-throughput sequencing). Next-generation sequencing has reduced the cost of DNA sequencing by at least five orders of magnitude, making it widely used in various fields of biology. According to sequencing principles, second-generation sequencing is usually divided into two categories: sequencing by synthesis and sequencing by ligation. The second-generation sequencing in this application is based on sequencing by synthesis. In the context of this specification, LNA is the English abbreviation of locked nucleic acid, which is called "locked nucleic acid" or "locked nucleic acid" in Chinese. LNA can combine with DNA or RNA to form hybrid oligonucleotide molecules. Therefore, the introduction of LNA can improve various technologies based on hybridization principles such as PCR reactions, microarray chips, and in situ hybridization. This type of nucleic acid can increase the melting problem of primers or probes, enhance the stability of these substances, and can be used in technologies such as real-time fluorescence quantitative PCR.
在本说明书的上下文中，cDNA是与细胞信使RNA(即mRNA)互补的DNA(complementary DNA)的克隆集合。而TSO全称是Template switch oligo，即模板转换寡核苷酸，是一种寡核苷酸，在反转录的过程中由逆转录酶加在非模板链5端的C寡核苷酸，用于下游的cDNA扩增。在第一链合成过程中，当到达RNA模板的5'末端时，MMLV逆转录酶的末端转移酶活性会在新合成的cDNA链的3'末端添加一些额外的核苷酸(主要是脱氧胞苷)。在本申请中，使用TSO用于配合逆转录酶启动转录组的逆转录。In the context of this specification, cDNA is a cloned collection of DNA (complementary DNA) that is complementary to cellular messenger RNA (i.e., mRNA). The full name of TSO is Template switch oligo, which is a template switch oligonucleotide. It is an oligonucleotide. During the reverse transcription process, the C oligonucleotide is added to the 5-end of the non-template strand by reverse transcriptase for Downstream cDNA amplification. During first-strand synthesis, when the 5' end of the RNA template is reached, the terminal transferase activity of MMLV reverse transcriptase adds some additional nucleotides (mainly deoxycytosine) to the 3' end of the newly synthesized cDNA strand. glycosides). In this application, TSO is used to cooperate with reverse transcriptase to initiate reverse transcription of the transcriptome.
在又一具体实施方式中，提供了上述方法，其中所述逆转录反应在甲基供体和金属盐存在下进行。在本说明书的上下文中，甲基供体是指在反应过程中提供甲基(-CH3)给受体的有关化合物。In yet another specific embodiment, the above method is provided, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt. In the context of this specification, methyl donor refers to a related compound that donates a methyl group (-CH3) to the acceptor during the reaction.
在再一具体实施方式中，提供了上述方法，其中所述甲基供体是甜菜碱。In yet another specific embodiment, the above method is provided, wherein the methyl donor is betaine.
在又一具体实施方式中，提供了上述方法，其中所述金属盐是镁盐。In yet another specific embodiment, the above method is provided, wherein the metal salt is a magnesium salt.
在再一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基以及0个或一个以上锁核酸(LNA)残基。In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide comprises one or two ribonucleotide residues and zero or more locked nucleic acid (LNA) residues.
在又一具体实施方式中，提供了上述方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。In yet another specific embodiment, the above method is provided, wherein the one or two ribonucleotide residues are riboguanine.
在再一具体实施方式中，提供了上述方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。 In yet another specific embodiment, the above method is provided, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
在又一具体实施方式中，提供了上述方法，其中所述锁核酸残基是锁鸟嘌呤。In yet another specific embodiment, the method above is provided, wherein the locked nucleic acid residue is locked guanine.
在再一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide comprises three nucleotide residues at the 3'-end characterized by the molecular formula rGrG+N, where +N represents locked Nucleotide residues.
在又一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸包含rGrG+G。In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide comprises rGrG+G.
在再一具体实施方式中，提供了上述方法，其中所述甲基供体是甜菜碱而所述金属盐是浓度至少为9mM的MgCl2。In yet another specific embodiment, there is provided the above method, wherein the methyl donor is betaine and the metal salt is MgCl2 at a concentration of at least 9mM.
在又一具体实施方式中，提供了上述方法，其中In yet another specific embodiment, the above method is provided, wherein
所述模板转换寡核苷酸选自由以下组成的组：The template switching oligonucleotide is selected from the group consisting of:
i.rGrG+G，优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，i.rGrG+G, preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N，ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and
iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
在本说明书的上下文中，rG和rC分别指作为核糖核酸的G和C在再一具体实施方式中，提供了上述方法，其中所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。In the context of this specification, rG and rC refer to G and C respectively as ribonucleic acid. In yet another specific embodiment, the above method is provided, wherein the cDNA is in a solution mixed with an oligonucleotide primer or in a It is synthesized from a colloidal polymer compound formed by polymerizing an oligonucleotide primer with a double bond and one or more compound monomers with a double bond.
在又一具体实施方式中，提供了上述方法，其中所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNN NNNN，其中“N”是任何核苷碱基，而“V”是“A”或“C”或“G”。In yet another specific embodiment, the above method is provided, wherein the oligonucleotide primer comprises SEQ ID NO. 5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNN NNNN, wherein "N" is any nucleoside base, and "V" is "A" or "C" or "G".
本申请在第四方面提供了分析多个单细胞中基因表达的方法。In a fourth aspect, the present application provides methods for analyzing gene expression in multiple single cells.
在一个具体实施方式中，提供了上述方法，其中该方法包括以下步骤：根据上述方法制备cDNA文库；并对cDNA文库进行测序。In a specific embodiment, the above method is provided, wherein the method includes the following steps: preparing a cDNA library according to the above method; and sequencing the cDNA library.
本申请在第五方面提供了一种模板转换寡核苷酸(TSO)。In a fifth aspect, the present application provides a template switching oligonucleotide (TSO).
在一个具体实施方式中，提供了上述TSO，其中在其3'-末端包含一个锁定的核苷酸残基。In a specific embodiment, a TSO as described above is provided which contains a locked nucleotide residue at its 3'-end.
在再一具体实施方式中，提供了上述TSO，其中其在3'-末端包含三个核苷酸残基，所述核苷酸残基选自+N+N+N、N+N+N、NN+N、rN+N+N和rNrN+N，其中每次出现的N独立地是脱氧核糖核苷酸残基，每次出现的rN独立地是核糖核苷酸残基，并且每次出现的+N独立地是锁定的核苷酸残基。 In yet another specific embodiment, there is provided the above TSO, wherein it comprises three nucleotide residues at the 3'-end, the nucleotide residues being selected from +N+N+N, N+N+N , NN+N, rN+N+N and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Occurrences of +N are independently locked nucleotide residues.
在又一具体实施方式中，提供了上述TSO，其中所述锁定核苷酸残基选自锁定的鸟嘌呤、锁定的腺嘌呤、锁定的尿嘧啶、锁定的胸腺嘧啶、锁定的胞嘧啶和锁定的5-甲基胞嘧啶。In yet another specific embodiment, the above-described TSO is provided, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine, and locked of 5-methylcytosine.
在再一具体实施方式中，提供了上述TSO，其中所述三个核苷酸残基选自NN+G、rNrN+G、GG+N、rGrG+G和GG+G。In yet another specific embodiment, there is provided a TSO as described above, wherein the three nucleotide residues are selected from the group consisting of NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
在一个具体实施方式中，提供了上述通过二代测序的建库方法，其中所述RNA是从选择性透过膜包围而成的反应隔室中的单细胞中裂解释放出的RNA。In a specific embodiment, the above-mentioned library construction method by second-generation sequencing is provided, wherein the RNA is RNA released from cleavage of single cells in a reaction compartment surrounded by a selectively permeable membrane.
在再一具体实施方式中，提供了上述方法，其中所述单细胞通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。In yet another specific embodiment, the above method is provided, wherein the single cell is packaged into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
在又一具体实施方式中，提供了上述方法，其中所述转座酶为Tn5转座酶。In yet another specific embodiment, the above method is provided, wherein the transposase is a Tn5 transposase.
在再一具体实施方式中，提供了上述方法，其中所述转座子末端结构域包括Tn5转座子末端结构域。In yet another specific embodiment, the above method is provided, wherein the transposon terminus domain comprises a Tn5 transposon terminus domain.
在又一具体实施方式中，提供了上述方法，其中所述方法进一步包括将所述第一双链产物cDNA与第二双链产物DNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。In yet another specific embodiment, the above method is provided, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product DNA to generate a pooled cDNA sample, and then labeling the pooled cDNA sample.
在再一具体实施方式中，提供了上述方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。In yet another specific embodiment, the above method is provided, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
在又一具体实施方式中，提供了上述方法，其中所述的方法都在选择性透过膜包围而成的反应隔室中进行。In yet another specific embodiment, the above method is provided, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
在一个具体实施方式中，提供了通过二代测序的建库方法，其中该方法包括：(a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；和通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于第一条DNA链延伸的条件下，与RNA分子互补，使其与TSO互补；In a specific embodiment, a library construction method by second-generation sequencing is provided, wherein the method includes: (a) formulating a composition including the following: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switching oligonucleotides; reverse transcriptase; and dNTPs; annealing a cDNA synthesis primer to an RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate; and converting an oligonucleotide by aligning the RNA-cDNA intermediate with a template The reverse transcriptase reaction is carried out by contacting with acid (TSO), where the TSO contains or does not contain a locked nucleic acid (LNA) at its 3'-end, which is complementary to the RNA molecule under conditions suitable for the elongation of the first DNA strand, making it Complementary to TSO;
(b)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物mRNA/cDNA杂交双链以产生标记样品；(b) hybridizing double strands with a transposome labeled product mRNA/cDNA comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
(c)将所述加接头DNA片段进行PCR扩增，得到扩增产物，(c) PCR amplify the adapter-added DNA fragment to obtain an amplification product,
RNA样品为上文涉及的所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA 捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物，其中(a)-(c)在上文涉及的反应隔室中进行。The RNA sample is the RNA to be analyzed and the RNA capture reagent mentioned above connected into a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA The capture reagent undergoes a biological or chemical reaction to generate the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after conversion, wherein (a)-(c) are performed in the reaction compartment referred to above.
在又一具体实施方式中，提供了上述方法，其中所述逆转录反应在甲基供体和金属盐存在下进行。In yet another specific embodiment, the above method is provided, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
在再一具体实施方式中，提供了上述方法，其中所述甲基供体是甜菜碱。In yet another specific embodiment, the above method is provided, wherein the methyl donor is betaine.
在又一具体实施方式中，提供了上述方法，其中所述金属盐是镁盐。In yet another specific embodiment, the above method is provided, wherein the metal salt is a magnesium salt.
在再一具体实施方式中，提供了上述方法，其中所述镁盐具有至少7mM的浓度。In yet another specific embodiment, the above method is provided, wherein the magnesium salt has a concentration of at least 7mM.
在又一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基和所述0个或一个以上锁核酸(LNA)残基。In yet another specific embodiment, there is provided the above method, wherein said template switching oligonucleotide comprises one or two ribonucleotide residues and said zero or more locked nucleic acid (LNA) residues.
在再一具体实施方式中，提供了上述方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。In yet another specific embodiment, the above-described method is provided, wherein the one or two ribonucleotide residues are riboguanine.
在又一具体实施方式中，提供了上述方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。In yet another specific embodiment, the above method is provided, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
在再一具体实施方式中，提供了上述方法，其中所述锁核酸残基是锁鸟嘌呤。In yet another specific embodiment, the method above is provided, wherein the locked nucleic acid residue is locked guanine.
在又一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide comprises three nucleotide residues at the 3'-end characterized by the formula rGrG+N, where +N represents locked Nucleotide residues.
在再一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸包含rGrG+G。In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide comprises rGrG+G.
在又一具体实施方式中，提供了上述方法，其中所述甲基供体是甜菜碱，而所述金属盐是浓度至少为9mM的MgCl2。In yet another specific embodiment, the above method is provided, wherein the methyl donor is betaine and the metal salt is MgCl2 at a concentration of at least 9mM.
在再一具体实施方式中，提供了上述方法，其中所述模板转换寡核苷酸选自由以下组成的组：In yet another specific embodiment, the above method is provided, wherein the template switching oligonucleotide is selected from the group consisting of:
i.rGrG+G，优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，i.rGrG+G, preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N，ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
在又一具体实施方式中，提供了上述方法，其中所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。In yet another specific embodiment, the above method is provided, wherein the cDNA is in a solution mixed with an oligonucleotide primer or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by polymerizing compound monomers.
在再一具体实施方式中，提供了上述方法，其中所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNNNNNN，其中“N” 是任何核苷碱基，而“V”是“A”或“C”或“G”。“T30”代表30个T。In yet another specific embodiment, the above method is provided, wherein the oligonucleotide primer comprises SEQ ID NO. 5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N" is any nucleobase, and "V" is "A" or "C" or "G". "T30" stands for 30 T's.
在又一具体实施方式中，提供了上述方法，其中该方法包括以下步骤：使用上述方法制备cDNA文库；并对cDNA文库进行测序。In yet another specific embodiment, the above method is provided, wherein the method includes the following steps: preparing a cDNA library using the above method; and sequencing the cDNA library.
在再一具体实施方式中，提供了上述方法，其中在其3'-最末端包含一个锁定的核苷酸残基。在又一具体实施方式中，提供了上述方法，其中所述RNA是选择性透过膜包围而成的反应隔室中的单细胞中裂解释放出的RNA。In yet another specific embodiment, the above method is provided, wherein a locked nucleotide residue is included at its 3'-most end. In yet another specific embodiment, the above method is provided, wherein the RNA is RNA released by cleavage in a single cell in a reaction compartment surrounded by a selectively permeable membrane.
在再一具体实施方式中，提供了上述方法，其中通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。In yet another specific embodiment, the above method is provided, wherein the microfluidic system is wrapped into a reaction compartment surrounded by a selectively permeable membrane.
在又一具体实施方式中，提供了上述方法，其中所述转座酶包括Tn5转座酶。In yet another specific embodiment, the above method is provided, wherein the transposase comprises Tn5 transposase.
在再一具体实施方式中，提供了上述方法，其中所述转座子末端结构域包括Tn5转座子末端结构域。In yet another specific embodiment, the above method is provided, wherein the transposon terminus domain comprises a Tn5 transposon terminus domain.
在又一具体实施方式中，提供了上述方法，其中所述方法进一步包括将第一双链产物cDNA与第二双链产物cDNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。In yet another specific embodiment, the above method is provided, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product cDNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
在再一具体实施方式中，提供了上述方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。In yet another specific embodiment, the above method is provided, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
在又一具体实施方式中，提供了上述本申请描述的方法，其中所述方法都在选择性透过膜包围而成的反应隔室中进行。In yet another specific embodiment, the method described in the above application is provided, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
实施例部分Example part
实施例1：单细胞mRNA捕获(打断DNA双链)Example 1: Single cell mRNA capture (breaking DNA double strands)
材料和试剂Materials and reagents
设备制造和操作。聚二甲基硅氧烷(PDMS)微流体装置使用所述的标准流程制造和操作(图3)。图3示出了产生含有双水相及靶向捕获试剂的液滴的微流体装置系统。Equipment manufacturing and operation. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated and operated using standard procedures as described (Figure 3). Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
双水相系统(ATPS，aqueous two-phase system)及靶向捕获试剂的制备。所有化学品均从Sigma-Aldrich和Fisher Scientific订购。使用APS(过硫酸铵)、10％(w/v)葡聚糖(MW 500K)、5’Acrydite poly T引物(带双键的寡核苷酸引物)SEQ ID NO.6：5`Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT)、5％(w/v)PEGDA(MW 8K)、5％(v/v)PEGDA(MW 575)、0.5％(w/v)制备双水相系统液滴(以下称为ATPS液滴)。可以使用其他浓度的PEGDA(MW 8K)和PEGDA(MW 575) 以及其他高分子聚合物。将含有上述所有成分的溶液混合在台式离心机中离心并诱导液-液相分离分别得到上层相即I相溶液和下层相即II相溶液，参见图3和图4。Preparation of aqueous two-phase system (ATPS, aqueous two-phase system) and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 5'Acrydite poly T primer (oligonucleotide primer with double bonds) SEQ ID NO.6: 5'Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTT ), 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) to prepare two-phase aqueous system droplets (hereinafter referred to as ATPS droplets) . Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) can be used and other polymers. The solution containing all the above components is mixed and centrifuged in a tabletop centrifuge to induce liquid-liquid phase separation to obtain the upper phase, i.e. phase I solution, and the lower phase, i.e. phase II solution, respectively, see Figures 3 and 4.
乳化(参见图3和图4)。对于较大尺寸的选择性透过膜包围而成的反应隔室的生成，如图1A和1B所示：液滴和选择性透过膜包围而成的反应隔室是使用50μm高度和40μm宽喷嘴的微流控芯片产生的(参见图3和图4)。使用的典型流速为：富含PEGD(M)A和5’Acrytite poly T引物的相(I相溶液)，流速为200μL/h，富含葡聚糖与细胞的混合液(II相溶液)，流速为100μL/h和液滴稳定油(载体油)(2％PEG-PFPE2HFE7500)，流速为600μL/h。通过图3所示的装置，如图4所示的过程就可以得到双水相系统液滴。Emulsification (see Figures 3 and 4). For the generation of reaction compartments surrounded by selectively permeable membranes of larger sizes, as shown in Figures 1A and 1B: the reaction compartment surrounded by droplets and selectively permeable membranes is made using a height of 50 μm and a width of 40 μm. The nozzle is produced by a microfluidic chip (see Figures 3 and 4). Typical flow rates used are: a phase rich in PEGD(M)A and 5'Acrytite poly T primers (Phase I solution), a flow rate of 200 μL/h, a mixture rich in dextran and cells (Phase II solution), The flow rate was 100 μL/h and the droplet stabilizing oil (carrier oil) (2% PEG-PFPE 2 HFE7500), the flow rate was 600 μL/h. Through the device shown in Figure 3, the two-phase aqueous system droplets can be obtained through the process shown in Figure 4.
交联(图5)。将双水相系统液滴收集在1.5ml管中，并使用高强度紫外线检查灯UVP(UVP,95-0127-01)在365nm波长下曝光2.5分钟立即交联(如图8所示，显示交联后成胶的状态)。5’Acrydite poly T引物和PEGDA壳硬化后，使用破乳剂(2％PEG-PFPE2的HFE7500)通过离心，从双水相系统液滴中回收所得选择性透过膜包围而成的反应隔室(图9)。Cross-linking (Figure 5). The two-phase aqueous system droplets were collected in a 1.5ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) exposed at 365nm wavelength for 2.5 minutes (as shown in Figure 8, showing cross-linking The state becomes glue after joining). After the 5'Acrydite poly T primer and PEGDA shell are hardened, a demulsifier (HFE7500 of 2% PEG-PFPE 2 ) is used to recover the reaction compartment surrounded by a selectively permeable membrane from the droplets of the two-phase aqueous system through centrifugation. (Figure 9).
细胞的裂解，rRNA和基因组DNA的去除。通过将选择性透过膜包围而成的反应隔室悬浮在含有以下物质的裂解缓冲液中进行所包裹细胞的裂解：200μg/mL蛋白酶K(Invitrogen,AM2546)、0.1％(v/v)Triton X-100(Sigma-Aldrich，T8787-100ML)、10mM Tris-HCl[pH 7.5]和1mM EDTA。将悬浮在裂解缓冲液中的选择性透过膜包围而成的反应隔室在37℃下孵育30分钟，然后在50℃下再孵育30分钟(如图12所示，显示了裂解后的状态)。Cell lysis, removal of rRNA and genomic DNA. Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 μg/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA. The reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 min and then at 50°C for an additional 30 min (as shown in Figure 12, showing the post-lysis state ).
在上述过程中，RNA被捕获试剂(硬化后的5’Acrydite poly T引物)捕获后，留存在反应隔室中(图2C和图10分别显示了反应隔室中的mRNA捕获的硬化后的带Poly T的寡核苷酸(引物)和细胞的概念图和实验结果图)。裂解后，将选择性透过膜包围而成的反应隔室进行rRNA的去除和基因组DNA的去除(图13)，提供两个试剂盒,即MGIEasy rRNA去除试剂盒(货号：MGI，1000005953)，DNase I(货号：Thermo Scientific，EN0521))，基于下表1-表6的配比和控温分别进行探针杂交、RNase H消化以及DNase I消化从而实现了rRNA的去除和基因组DNA的去除，在每次操作过程中在上一步操作结束后需要对选择性透过膜包围而成的反应隔室进行离心清洗。如图13所示，在荧光显微镜下没有荧光，显示基因组DNA已经被除去。During the above process, RNA is captured by the capture reagent (hardened 5'Acrydite poly T primer) and remains in the reaction compartment (Figure 2C and Figure 10 respectively show the hardened bands captured by mRNA in the reaction compartment). Poly T oligonucleotides (primers) and cells (conceptual diagram and experimental results diagram). After lysis, the reaction compartment surrounded by a selective permeable membrane is used to remove rRNA and genomic DNA (Figure 13). Two kits are provided, namely MGIEasy rRNA Removal Kit (Cat. No.: MGI, 1000005953). DNase I (Cat. No.: Thermo Scientific, EN0521)), based on the ratio and temperature control in Table 1-Table 6 below, perform probe hybridization, RNase H digestion and DNase I digestion respectively to achieve the removal of rRNA and genomic DNA. During each operation, the reaction compartment surrounded by the selectively permeable membrane needs to be centrifuged and cleaned after the previous step. As shown in Figure 13, there is no fluorescence under a fluorescence microscope, indicating that the genomic DNA has been removed.
探针杂交所用到的相关实验参数： Relevant experimental parameters used for probe hybridization:
表1在冰上配制杂交反应混合液
Table 1 Preparing hybridization reaction mixture on ice
表2杂交反应的温控程序
Table 2 Temperature control program for hybridization reaction
表3 RNase H消化：
Table 3 RNase H digestion:
表4 RNase H消化的温控程序
Table 4 Temperature control program for RNase H digestion
表5 DNase I消化：

Table 5 DNase I digestion:

表6 DNase I消化的温控程序
Table 6 Temperature control program for DNase I digestion
mRNA逆转录及扩增。将上一步得到的选择性透过膜包围而成的反应隔室悬浮在含有0.5U/μL Superscript IV、1X First Strand Buffer的逆转录酶中，然后在50℃下再孵育30分钟。类似宏观反应的PCR用于扩增cDNA或特异基因的特定区域片段。使用KAPA PCR试剂盒(KAPABiosystems,KK2602)进行35个循环的每次扩增。在所有酶促反应中，选择性透过膜包围而成的反应隔室大约占据了最终反应体积的40-50％(图14、图15、图16和图17)。图14显示选择性透过膜组成的反应隔室内进行单细胞mRNA反转录cDNA扩增的荧光染色；图15显示选择性透过膜组成的反应隔室内单细胞mRNA反转录cDNA扩增的结果；图16显示选择性透过膜组成的反应隔室内单细胞mRNA反转录cDNA扩增的Qsep表征；图17选择性透过膜组成的反应隔室内进行单细胞mRNA反转录cDNA扩增，进行流式分析和分选。mRNA reverse transcription and amplification. Suspend the reaction compartment surrounded by the selectively permeable membrane obtained in the previous step in reverse transcriptase containing 0.5U/μL Superscript IV and 1X First Strand Buffer, and then incubate at 50°C for another 30 minutes. PCR, which is similar to a macro reaction, is used to amplify fragments of specific regions of cDNA or specific genes. Each amplification was performed for 35 cycles using KAPA PCR kit (KAPA Biosystems, KK2602). In all enzymatic reactions, the reaction compartment surrounded by the selectively permeable membrane occupies approximately 40-50% of the final reaction volume (Figure 14, Figure 15, Figure 16 and Figure 17). Figure 14 shows fluorescent staining of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane; Figure 15 shows single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane. Results; Figure 16 shows Qsep characterization of single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane; Figure 17 Single-cell mRNA reverse transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane , for flow analysis and sorting.
扩增产物的打断及捕获。使用DNA打断试剂盒(NexteraXT DNA Library Preparation Kit(24 samples),FC-131-1024)对cDNA扩增产物进行打断。使用Tn5转座酶二聚体(Tn5-Adaptor)作为捕获试剂可将短片段DNA(片段化的核酸)维持在多聚体状态，留存在由选择性透过膜包围而成的反应隔室中(图2A显示了整个过程的示意图，图18显示了cDNA扩增文库被打断，图19cDNA扩增文库打断的Qsep表征)。以用于后续的单细胞测序。Interruption and capture of amplification products. Use DNA fragmentation kit (NexteraXT DNA Library Preparation Kit (24 samples), FC-131-1024) to fragment the cDNA amplification product. Using Tn5 transposase dimer (Tn5-Adaptor) as a capture reagent can maintain short fragments of DNA (fragmented nucleic acids) in a polymeric state and retain them in a reaction compartment surrounded by a selectively permeable membrane. (Figure 2A shows a schematic diagram of the entire process, Figure 18 shows the fragmentation of the cDNA amplified library, and Figure 19 shows the Qsep characterization of fragmentation of the cDNA amplification library). for subsequent single-cell sequencing.
实施例2：单细胞mRNA捕获(打断RNA/DNA杂交链，即没有做PCR扩增，直接逆转录后打断的实施例)Example 2: Single cell mRNA capture (interruption of RNA/DNA hybrid chain, that is, no PCR amplification, direct reverse transcription and then interruption)
材料和试剂Materials and reagents
设备制造和操作。聚二甲基硅氧烷(PDMS)微流体装置使用所述的标准流程制造和操作(图3)。图3示出了产生含有双水相及靶向捕获试剂的液滴的微流体装置系统。Equipment manufacturing and operation. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated and operated using standard procedures as described (Figure 3). Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
ATPS及靶向捕获试剂的制备。所有化学品均从Sigma-Aldrich和Fisher Scientific订购。使用APS(过硫酸铵)、10％(w/v)葡聚糖(MW 500K)、5’Acrytide poly T引物： 例如可以使用SEQ ID NO.6：5`Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT、5％(w/v)PEGDA(MW 8K)、5％(v/v)PEGDA(MW 575)、0.5％(w/v)制备ATPS液滴(以下均称为ATPS液滴)。可以使用其他浓度的PEGDA(MW 8K)和PEGDA(MW 575)以及其他高分子聚合物。将含有上述所有成分的溶液混合并在台式离心机中诱导液-液相分离分别得到上层相即I相溶液和下层相即II相溶液。Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 5'Acrytide poly T primer: For example, SEQ ID NO. 6: 5'Acrydite AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) can be used to prepare ATPS liquid droplets (hereinafter referred to as ATPS droplets). Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. The solutions containing all the above components are mixed and liquid-liquid phase separation is induced in a tabletop centrifuge to obtain the upper phase, i.e., phase I solution, and the lower phase, i.e., phase II solution.
乳化(图4)。对于较大尺寸的选择性透过膜包围而成的反应隔室生成，如图1A和1B所示：液滴和选择性透过膜包围而成的反应隔室是使用50μm高度和40μm宽喷嘴的微流控芯片产生的。使用的典型流速为：富含PEGD(M)A的相(I相溶液)，流速为200μL/h，富含葡聚糖与细胞的相(II相溶液)，流速为100μL/h和液滴稳定油(载体油)(2％PEG-PFPE2HFE7500)，流速为600μL/h。Emulsification (Figure 4). For the generation of reaction compartments surrounded by selectively permeable membranes of larger sizes, as shown in Figures 1A and 1B: the reaction compartments surrounded by droplets and selectively permeable membranes are generated using 50 μm height and 40 μm wide nozzles. of microfluidic chips produced. Typical flow rates used are: PEGD(M)A-rich phase (phase I solution) with a flow rate of 200 μL/h, dextran and cell-rich phase (phase II solution) with a flow rate of 100 μL/h and droplets Stabilizing oil (carrier oil) (2% PEG-PFPE 2 HFE7500), flow rate 600 μL/h.
由于双相系统的粘度增加，可以观察到喷射机制造成的液滴破裂，这可以通过调整系统的流速转变液滴生成模式。在此，图4示出了通过微流体装置系统向微流控芯片中注入第一种流体(I相溶液，富含葡聚糖)、第二种流体(II相溶液，富含基于聚乙二醇的聚合物)、连续相(载体油为氟化油并且包含表面活性剂，例如PFPE-PEG-PFPE(全氟聚醚-聚乙二醇-全氟聚醚)三嵌段共聚物)；靶向捕获试剂从第一种流体、第二种流体、连续相(载体油)或任意两者或三者进入微流控体系。As the viscosity of the biphasic system increases, droplet breakup by the jetting mechanism can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system. Here, Figure 4 shows the injection of the first fluid (Phase I solution, rich in dextran), the second fluid (Phase II solution, rich in polyethylene-based) into the microfluidic chip through the microfluidic device system. glycol), continuous phase (the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer) ;The target capture reagent enters the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.
交联(图5)。将乳液收集在1.5ml管中，并使用高强度紫外线检查灯UVP(UVP,95-0127-01)在365nm波长下曝光2.5分钟立即交联(图8，显示交联后成胶的状态)。PEGDA壳硬化后，使用破乳剂(2％PEG-PFPE2的HFE7500)从乳液中回收所得选择性透过膜包围而成的反应隔室(图9)。在此，图5示出了通过引发聚合来硬化外层II相的方法。图8示出了成胶后的选择性透过膜包围而成的反应隔室。图9示出了破乳后的选择性透过膜包围而成的反应隔室。Cross-linking (Figure 5). The emulsion was collected in a 1.5 ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) at a wavelength of 365 nm for 2.5 minutes (Figure 8, showing the gel state after cross-linking). After the PEGDA shell hardened, a demulsifier (2% PEG-PFPE 2 in HFE7500) was used to recover the reaction compartment surrounded by a selectively permeable membrane from the emulsion (Figure 9). Here, FIG. 5 shows a method for hardening the outer layer II phase by initiating polymerization. Figure 8 shows the reaction compartment surrounded by the selectively permeable membrane after gelation. Figure 9 shows a reaction compartment surrounded by a selectively permeable membrane after demulsification.
细胞的裂解，rRNA基因组DNA的去除。通过将选择性透过膜包围而成的反应隔室悬浮在含有以下物质的裂解缓冲液中进行所包裹细胞的裂解：200μg/mL蛋白酶K(Invitrogen,AM2546)、0.1％(v/v)Triton X-100(Sigma-Aldrich，T8787-100ML)、10mM Tris-HCl[pH 7.5]和1mM EDTA。将悬浮在裂解缓冲液中的选择性透过膜包围而成的反应隔室在37℃下孵育30分钟，然后在50℃下再孵育30分钟(图12，显示了裂解后的状态)。在此，图12示出了选择性透过膜包围而成的反应隔室内进行细胞裂解。Lysis of cells, removal of rRNA and genomic DNA. Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 μg/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA. The reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 minutes and then at 50°C for an additional 30 minutes (Figure 12, showing the post-lysis state). Here, FIG. 12 shows cell lysis in a reaction compartment surrounded by a selectively permeable membrane.
在此过程中，RNA被捕获试剂(Acrytide polyT引物)捕获后，留存在反应隔室中(图 2C，图10.反应隔室中的mRNA捕获的带双键的寡核苷酸(引物)和细胞)。裂解后，将选择性透过膜包围而成的反应隔室进行rRNA的去除和基因组DNA的去除(图13)。在此，图13示出了选择性透过膜包围而成的反应隔室内进行去除基因组DNA。During this process, RNA is captured by the capture reagent (Acrytide polyT primer) and remains in the reaction compartment (Figure 2C, Figure 10. Double-bonded oligonucleotides (primers and cells) captured by mRNA in the reaction compartment. After lysis, rRNA removal and genomic DNA removal are performed in a reaction compartment surrounded by a selectively permeable membrane (Figure 13). Here, Figure 13 shows that genomic DNA is removed in a reaction compartment surrounded by a selectively permeable membrane.
探针杂交所用到的相关实验参数：Relevant experimental parameters used for probe hybridization:
表7在冰上配制杂交反应混合液
Table 7 Preparing hybridization reaction mixture on ice
表8杂交反应的温控程序
Table 8 Temperature control program for hybridization reaction
表9.RNase H消化：
Table 9. RNase H digestion:
表10 RNase H消化的温控程序
Table 10 Temperature control program for RNase H digestion
表11.DNase I消化：

Table 11. DNase I digestion:

表12 DNase I消化的温控程序
Table 12 Temperature control program for DNase I digestion
mRNA逆转录及打断。通过将选择性透过膜包围而成的反应隔室悬浮在含有0.5U/μL Superscript IV、1X First Strand Buffer的逆转录酶中，然后在50℃下再孵育30分钟。使用DNA打断试剂盒(Nextera XT DNA Library Preparation Kit(24 samples),FC-131-1024)对扩增产物RNA/DNA杂交链进行打断。使用Tn5二聚体作为捕获试剂可将短片段RNA/DNA杂交链维持在多聚体状态，留存在由选择性透过膜包围而成的反应隔室中(图2B、图20显示RNA/DNA杂交链的打断和图21)。在此，图20示出了选择性透过膜包围而成的反应隔室内的RNA/DNA杂交链打断。Reverse transcription and interruption of mRNA. The reaction compartment surrounded by a selectively permeable membrane is suspended in reverse transcriptase containing 0.5U/μL Superscript IV, 1X First Strand Buffer, and then incubated at 50°C for an additional 30 minutes. Use DNA fragmentation kit (Nextera XT DNA Library Preparation Kit (24 samples), FC-131-1024) to fragment the RNA/DNA hybrid chain of the amplified product. Using Tn5 dimers as capture reagents can maintain short fragments of RNA/DNA hybrid chains in a polymeric state and remain in the reaction compartment surrounded by a selectively permeable membrane (Figure 2B, Figure 20 shows RNA/DNA Interruption of hybridized chains and Figure 21). Here, FIG. 20 shows the interruption of RNA/DNA hybrid chains in a reaction compartment surrounded by a selectively permeable membrane.
而图21示出了选择性透过膜包围而成的反应隔室内的RNA/DNA杂交链打断的Bioanalzyer 2100表征。Figure 21 shows the Bioanalzyer 2100 characterization of RNA/DNA hybrid chain interruption in a reaction compartment surrounded by a selective permeable membrane.
实施例3：单细胞mRNA捕获(磁珠拓扑捕获，打断DNA双链)Example 3: Single-cell mRNA capture (magnetic bead topological capture, breaking DNA double strands)
材料和试剂Materials and reagents
设备制造和操作。聚二甲基硅氧烷(PDMS)微流体装置使用所述的标准流程制造和操作(图3)。图3示出了产生含有双水相及靶向捕获试剂的液滴的微流体装置系统。Equipment manufacturing and operation. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated and operated using standard procedures as described (Figure 3). Figure 3 shows a microfluidic device system that generates droplets containing an aqueous dual phase and targeted capture reagents.
ATPS及靶向捕获试剂的制备。所有化学品均从Sigma-Aldrich和Fisher Scientific订购。使用APS(过硫酸铵)、10％(w/v)葡聚糖(MW 500K)、5’biotinylatedpolyT引物、链霉亲和素磁珠、5％(w/v)PEGDA(MW 8K)、5％(v/v)PEGDA(MW 575)、0.5％(w/v)制备ATPS液滴。可以使用其他浓度的PEGDA(MW 8K)和PEGDA(MW 575)以及其他高分子聚合物。将含有所有成分的溶液混合并在台式离心机中诱导液-液相分离。Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 5' biotinylated polyT primer, streptavidin magnetic beads, 5% (w/v) PEGDA (MW 8K), 5 % (v/v) PEGDA (MW 575), 0.5% (w/v) to prepare ATPS droplets. Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. The solution containing all components was mixed and liquid-liquid phase separation was induced in a tabletop centrifuge.
链霉亲和素磁珠偶联5’biotinylated polyT引物。通过将磁珠悬浮在含有以下物质的缓冲液中进行5’biotinylatedpolyT的偶联：10mM Tris-HCl(pH 7.5)，1mM EDTA，1 M NaCl，0.05％Tween-20，1uM 5’biotinylated polyT。置于旋转混合仪上，室温旋转混合30min，制备得到捕获试剂。Streptavidin magnetic beads coupled 5' biotinylated polyT primer. Coupling of 5'biotinylatedpolyT was performed by suspending magnetic beads in buffer containing: 10mM Tris-HCl (pH 7.5), 1mM EDTA, 1 M NaCl, 0.05% Tween-20, 1uM 5'biotinylated polyT. Place it on a rotating mixer and rotate and mix at room temperature for 30 minutes to prepare the capture reagent.
乳化(图4)。对于较大尺寸的选择性透过膜包围而成的反应隔室生成，如图所1所示：液滴和选择性透过膜包围而成的反应隔室是使用50μm高度和40μm宽喷嘴的微流控芯片产生的。使用的典型流速为：富含PEGD(M)A的I相溶液-流速为200μL/h，富含葡聚糖、细胞、捕获试剂的II相溶液-流速为100μL/h和液滴稳定油(2％PEG-PFPE2HFE7500)-流速为600μL/h。由于双相系统的粘度增加，可以观察到喷射机制造成的液滴破裂，这可以通过调整系统的流速转变液滴生成模式(图4和图22)。在此，图4示出了通过微流体装置系统向微流控芯片中注入第一种流体(I相溶液，富含葡聚糖)、第二种流体(II相溶液，富含基于聚乙二醇的聚合物)、连续相(载体油为氟化油并且包含表面活性剂，例如PFPE-PEG-PFPE(全氟聚醚-聚乙二醇-全氟聚醚)三嵌段共聚物)；靶向捕获试剂从第一种流体、第二种流体、连续相(载体油)或任意两者或三者进入微流控体系。在此，图22示出了选择性透过膜组成的反应隔室成胶前的状态(磁珠拓扑捕获)。Emulsification (Figure 4). For the generation of a reaction compartment surrounded by a selectively permeable membrane of larger size, as shown in Figure 1: the reaction compartment surrounded by droplets and selectively permeable membranes uses a 50μm height and 40μm wide nozzle Produced by microfluidic chips. Typical flow rates used are: Phase I solution rich in PEGD(M)A - flow rate 200 μL/h, Phase II solution rich in dextran, cells, capture reagent - flow rate 100 μL/h and droplet stabilizing oil ( 2% PEG-PFPE 2 HFE7500) - flow rate 600 μL/h. As the viscosity of the biphasic system increases, droplet breakup by the jet can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system (Figures 4 and 22). Here, Figure 4 shows the injection of the first fluid (Phase I solution, rich in dextran), the second fluid (Phase II solution, rich in polyethylene-based) into the microfluidic chip through the microfluidic device system. glycol), continuous phase (the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer) ;The target capture reagent enters the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three. Here, Figure 22 shows the state of the reaction compartment composed of a selectively permeable membrane before gelation (magnetic bead topological capture).
交联(图5)。将乳液收集在1.5ml管中，并使用高强度紫外线检查灯UVP(UVP,95-0127-01)在365nm波长下曝光2.5分钟立即交联(图23)。PEGDA壳硬化后，使用破乳剂(2％PEG-PFPE2的HFE7500)从乳液中回收所得选择性透过膜包围而成的反应隔室(图24)。在此，图5示出了通过引发聚合来硬化外层II相的方法。在此，图23示出了选择性透过膜组成的反应隔室成胶后的状态(磁珠拓扑捕获)。在此，图24示出了选择性透过膜组成的反应隔室破乳后(磁珠拓扑捕获)。Cross-linking (Figure 5). The emulsion was collected in a 1.5 ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) exposed at 365 nm wavelength for 2.5 minutes (Figure 23). After the PEGDA shell hardened, a demulsifier (HFE7500 of 2% PEG-PFPE 2 ) was used to recover the reaction compartment surrounded by a selectively permeable membrane from the emulsion (Figure 24). Here, FIG. 5 shows a method for hardening the outer layer II phase by initiating polymerization. Here, Figure 23 shows the state of the reaction compartment composed of a selectively permeable membrane after gelation (magnetic bead topological capture). Here, Figure 24 shows a reaction compartment composed of a selectively permeable membrane after demulsification (magnetic bead topological capture).
细胞的裂解，rRNA基因组DNA的去除。通过将选择性透过膜包围而成的反应隔室悬浮在含有以下物质的裂解缓冲液中进行所包裹细胞的裂解：200μg/mL蛋白酶K(Invitrogen,AM2546)、0.1％(v/v)Triton X-100(Sigma-Aldrich，T8787-100ML)、10mM Tris-HCl[pH 7.5]和1mM EDTA。将悬浮在裂解缓冲液中的选择性透过膜包围而成的反应隔室在37℃下孵育30分钟，然后在50℃下再孵育30分钟(图25)。在此，图25示出了在选择性透过膜组成的反应隔室内进行细胞裂解(磁珠拓扑捕获)。Lysis of cells, removal of rRNA and genomic DNA. Lysis of the enclosed cells was performed by suspending the reaction compartment surrounded by a selectively permeable membrane in a lysis buffer containing: 200 μg/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA. The reaction compartment surrounded by a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 minutes and then at 50°C for an additional 30 minutes (Figure 25). Here, Figure 25 shows cell lysis (magnetic bead topological capture) in a reaction compartment consisting of a selectively permeable membrane.
在过程中，RNA被链霉亲和素磁珠捕获后，留存在反应隔室中。During the process, RNA is captured by streptavidin magnetic beads and remains in the reaction compartment.
裂解后，将选择性透过膜包围而成的反应隔室进行rRNA的去除和基因组DNA的去除(图26)。在此，在图26中示出了选择性透过膜组成的反应隔室内进行去除基因组DNA(磁珠拓扑捕获)。After lysis, rRNA removal and genomic DNA removal are performed in a reaction compartment surrounded by a selectively permeable membrane (Figure 26). Here, removal of genomic DNA (magnetic bead topological capture) is performed within a reaction compartment consisting of a selectively permeable membrane shown in Figure 26 .
探针杂交所用到的相关实验参数： Relevant experimental parameters used for probe hybridization:
表13在冰上配制杂交反应混合液
Table 13 Preparing hybridization reaction mixture on ice
表14杂交反应的温控程序
Table 14 Temperature control program for hybridization reaction
表15.RNase H消化：
Table 15. RNase H digestion:
表16 RNase H消化的温控程序
Table 16 Temperature control program for RNase H digestion
表17.DNase I消化：
Table 17. DNase I digestion:
表18 DNase I消化的温控程序
Table 18 Temperature control program for DNase I digestion
mRNA逆转录及扩增。通过将选择性透过膜包围而成的反应隔室悬浮在含有0.5U/μL Superscript IV、1X First Strand Buffer的逆转录酶中，然后在50℃下再孵育30分钟。类似宏观反应的PCR用于扩增cDNA或特异基因的特定区域片段。根据制造商的建议，使用KAPA PCR试剂盒(KAPABiosystems,KK2602)进行35个循环的每次扩增。在所有酶促反应中，选择性透过膜包围而成的反应隔室大约占据了最终反应体积的40-50％(图27)。图27示出了在选择性透过膜组成的反应隔室内进行单细胞mRNA反转录cDNA扩增的荧光染色(磁珠拓扑捕获)。mRNA reverse transcription and amplification. The reaction compartment surrounded by a selectively permeable membrane is suspended in reverse transcriptase containing 0.5U/μL Superscript IV, 1X First Strand Buffer, and then incubated at 50°C for an additional 30 minutes. PCR, which is similar to a macro reaction, is used to amplify fragments of specific regions of cDNA or specific genes. Each amplification was performed for 35 cycles using the KAPA PCR kit (KAPA Biosystems, KK2602) according to the manufacturer's recommendations. In all enzymatic reactions, the reaction compartment surrounded by a selectively permeable membrane occupies approximately 40-50% of the final reaction volume (Figure 27). Figure 27 shows fluorescent staining (magnetic bead topological capture) of single-cell mRNA reverse-transcription cDNA amplification in a reaction compartment composed of a selectively permeable membrane.
扩增产物的打断及捕获。根据制造商的建议，使用DNA打断试剂盒(Nextera XT DNA Library PreparationKit(24samples),FC-131-1024)对扩增产物进行打断。使用Tn5二聚体作为捕获试剂可将短片段DNA维持在多聚体状态，留存在由选择性透过膜包围而成的反应隔室中。Interruption and capture of amplification products. The amplified products were fragmented using the DNA fragmentation kit (Nextera XT DNA Library PreparationKit (24samples), FC-131-1024) according to the manufacturer's recommendations. Using Tn5 dimers as capture reagents can maintain short fragments of DNA in a polymeric state in a reaction compartment surrounded by a selectively permeable membrane.
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本发明保护之列。 Although the embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive. . Under the inspiration of this description and without departing from the scope of protection of the claims of the present invention, those of ordinary skill in the art can also make many forms, which are all included in the protection of the present invention.

Claims (62)

  1. 一种基于拓扑捕获的RNA样本处理系统，其包括：An RNA sample processing system based on topological capture, which includes:
    a)作为反应隔室外层的选择性透过膜，所述选择性透过膜能够选择性地透过RNA样本处理试剂；a) As a selectively permeable membrane on the outer layer of the reaction compartment, the selectively permeable membrane can selectively pass through the RNA sample processing reagent;
    b)位于所述反应隔室内部的内容物，内容物包括RNA捕获试剂和待分析RNA；其中，b) Contents located inside the reaction compartment, including RNA capture reagents and RNA to be analyzed; wherein,
    所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物；The RNA to be analyzed and the RNA capture reagent are connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed and the RNA capture reagent are transformed through biological or chemical reactions. The nucleic acid content of the formed deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA);
    所述选择性透过膜能够选择性地留存RNA捕获试剂和待分析RNA连接而成的整体或复合物或核酸内容物；The selectively permeable membrane can selectively retain the whole or complex or nucleic acid content formed by connecting the RNA capture reagent and the RNA to be analyzed;
    所述整体或复合物或核酸内容物的直径大于所述选择性透过膜的膜孔的孔径的1/2。The diameter of the whole or complex or nucleic acid content is greater than 1/2 the pore size of the membrane pores of the selectively permeable membrane.
  2. 根据权利要求1所述的基于拓扑捕获的RNA样本处理系统，其中，The RNA sample processing system based on topological capture according to claim 1, wherein,
    位于所述反应隔室内部的RNA捕获试剂、待分析RNA，以及所述RNA捕获试剂和待分析RNA连接而成的整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物为液态、胶状或半液态；RNA capture reagent, RNA to be analyzed located inside the reaction compartment, and the whole or complex formed by connecting the RNA capture reagent and the RNA to be analyzed, or the deoxyribonucleic acid (DNA) and/or the deoxyribonucleic acid (DNA) formed after the conversion or the nucleic acid content of ribonucleic acid (RNA) is liquid, gelatinous or semi-liquid;
    所述反应隔室内部还包含渗透压调节剂；The reaction compartment also contains an osmotic pressure regulator inside;
    优选所述渗透压调节剂是葡聚糖。Preferably the osmotic pressure regulator is dextran.
  3. 根据权利要求1～2中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，The RNA sample processing system based on topological capture according to any one of claims 1 to 2, wherein,
    所述捕获试剂为由带双键的寡核苷酸引物自身聚合而成的胶状高分子化合物或带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物；The capture reagent is a colloidal polymer compound formed by the polymerization of an oligonucleotide primer with a double bond itself or a polymerization of an oligonucleotide primer with a double bond and one or more compound monomers with a double bond. Colloidal polymer compound formed;
    所述RNA捕获试剂与所述待分析RNA进行逆转录反应得到直径大于选择性透过膜孔径的1/2的产物分子。The RNA capture reagent performs a reverse transcription reaction with the RNA to be analyzed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  4. 根据权利要求3所述的基于拓扑捕获的RNA样本处理系统，其中，The RNA sample processing system based on topological capture according to claim 3, wherein,
    所述RNA捕获试剂与所述待分析RNA进行逆转录反应后，进行PCR扩增反应，得到直径大于选择性透过膜孔径的1/2的产物分子。 After performing a reverse transcription reaction between the RNA capture reagent and the RNA to be analyzed, a PCR amplification reaction is performed to obtain product molecules with a diameter greater than 1/2 of the pore size of the selectively permeable membrane.
  5. 根据权利要求1～4中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，The RNA sample processing system based on topological capture according to any one of claims 1 to 4, wherein,
    所述待分析RNA，以及所述RNA捕获试剂和待分析RNA连接成整体或复合物，或所述转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物后，进一步地通过以下的第二捕获试剂进行捕获：After the RNA to be analyzed, the RNA capture reagent and the RNA to be analyzed are connected into a whole or a complex, or the nucleic acid content of the deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after the conversion, Further capture is performed with the following second capture reagent:
    所述第二捕获试剂为DNA转座酶和DNA的复合物；The second capture reagent is a complex of DNA transposase and DNA;
    优选所述DNA转座酶为Tn5，所述DNA在其末端具有转座酶识别序列。Preferably, the DNA transposase is Tn5, and the DNA has a transposase recognition sequence at its end.
  6. 根据权利要求1～5中任一项所述的基于拓扑捕获的RNA样本处理系统，其中，The RNA sample processing system based on topological capture according to any one of claims 1 to 5, wherein,
    所述待分析RNA来源于同一个细胞或细胞核；优选所述待分析RNA处于完整的细胞或细胞核中。The RNA to be analyzed originates from the same cell or nucleus; preferably, the RNA to be analyzed is in a complete cell or nucleus.
  7. 制造根据权利要求1至6的任一项所述的基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：The method of manufacturing the RNA sample processing system based on topological capture according to any one of claims 1 to 6, which includes the following steps:
    准备包括透压调节剂和第一水性溶剂的第一相；preparing a first phase including a tonicity regulator and a first aqueous solvent;
    准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;
    RNA捕获试剂添加在第一相或第二相中；RNA capture reagent is added in the first or second phase;
    将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；Mix the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
    将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；以及Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and
    对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；The above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
    对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室；Demulsifying the water-in-oil emulsion that has been cured or semi-cured to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the interior;
    将外层具有选择性透过膜、在内部具有内容物的反应隔室混合至细胞裂解液以释放细胞或细胞核内的RNA或经过加热释放细胞或细胞核内的RNA从而与RNA捕获试剂接触。A reaction compartment with a selectively permeable membrane on the outside and content on the inside is mixed into a cell lysate to release RNA in cells or nuclei or is heated to release RNA in cells or nuclei to be contacted with an RNA capture reagent.
  8. 制造基于拓扑捕获的RNA样本处理系统的方法，其包括如下步骤：A method of manufacturing an RNA sample processing system based on topological capture, which includes the following steps:
    准备包括RNA捕获试剂、渗透压调节剂和第一水性溶剂的第一相； preparing a first phase including an RNA capture reagent, a tonicity regulator, and a first aqueous solvent;
    准备混合有选择性透过膜形成材料和第二水性溶剂的第二相；preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;
    将处于完整的细胞或细胞核中的待分析RNA混合至第一相或第二相；优选将处于完整的细胞或细胞核中的待分析RNA混合至第一相；Mix the RNA to be analyzed in intact cells or cell nuclei into the first phase or the second phase; preferably, mix the RNA to be analyzed in intact cells or cell nuclei into the first phase;
    将细胞裂解液混合至第一相或第二相；优选细胞裂解液与细胞或细胞核在不同的相；Mix the cell lysate into the first phase or the second phase; preferably the cell lysate and the cells or cell nuclei are in different phases;
    将第一相和第二相混合为混合亲水相，再将所述混合亲水相与所述油性溶剂混合，制得油包水乳剂；且细胞裂解液辅助释放细胞或细胞核内的RNA从而与RNA捕获试剂接触；以及Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with the oily solvent to prepare a water-in-oil emulsion; and the cell lysis solution assists in releasing RNA in the cells or cell nuclei to thereby Contact with RNA capture reagents; and
    对上述油包水乳剂进行固化或半固化反应形成选择性透过膜；The above water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane;
    对进行了固化或半固化反应后的所述油包水乳剂破乳，得到在外层具有选择性透过膜、在内部具有内容物的反应隔室。The water-in-oil emulsion that has been cured or semi-cured is demulsified to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the inside.
  9. 利用根据权利要求1至6的任一项所述的基于拓扑捕获的RNA样本处理系统制备二代测序(NGS)文库的方法，该方法包括：A method for preparing a next-generation sequencing (NGS) library using the topological capture-based RNA sample processing system according to any one of claims 1 to 6, the method comprising:
    (a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；(a) Preparing a composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
    将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；并且通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于所述第一条cDNA链延伸的条件下，与RNA分子互补，使其与TSO互补；Annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and performing a reverse transcriptase reaction by contacting the RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein The TSO may or may not contain a locked nucleic acid (LNA) at its 3'-end that is complementary to the RNA molecule under conditions suitable for extension of the first cDNA strand, making it complementary to the TSO;
    (b)在足以产生产物双链DNA的扩增条件下，对RNA-cDNA中间体进行二链合成扩增，得到第二条DNA；(b) Under amplification conditions sufficient to produce the product double-stranded DNA, perform double-stranded synthetic amplification of the RNA-cDNA intermediate to obtain the second DNA;
    (c)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物第二条DNA以产生标记样品；(c) labeling the product with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
    (d)将所述加接头DNA片段进行PCR扩增，得到扩增产物；(d) PCR amplify the adapter-added DNA fragment to obtain an amplification product;
    RNA样品为权利要求1～6中任一项中涉及的所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物，The RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of claims 1 to 6 connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed is RNA and the RNA capture reagent undergo a biological or chemical reaction to generate the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after conversion,
    其中(a)-(d)在权利要求1～6中任一项中涉及的反应隔室中进行。 Wherein (a)-(d) are carried out in the reaction chamber according to any one of claims 1 to 6.
  10. 如权利要求9所述的方法，其中，所述逆转录反应在甲基供体和金属盐存在下进行。The method of claim 9, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
  11. 如权利要求11所述的方法，其特征在于，所述甲基供体是甜菜碱。The method of claim 11, wherein the methyl donor is betaine.
  12. 如权利要求11所述的方法，其特征在于，所述金属盐是镁盐。The method of claim 11, wherein the metal salt is a magnesium salt.
  13. 如权利要求12所述的方法，其中所述镁盐具有7mM以上的浓度。The method of claim 12, wherein the magnesium salt has a concentration above 7mM.
  14. 如权利要求9所述的方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基以及0个或一个以上锁核酸(LNA)残基。The method of claim 9, wherein the template switching oligonucleotide comprises one or two ribonucleotide residues and zero or more locked nucleic acid (LNA) residues.
  15. 如权利要求14所述的方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。The method of claim 14, wherein the one or two ribonucleotide residues are riboguanine.
  16. 如权利要求14所述的方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。The method of claim 14, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  17. 如权利要求16所述的方法，其特征在于，所述锁核酸残基是锁鸟嘌呤。The method of claim 16, wherein the locked nucleic acid residue is locked guanine.
  18. 如权利要求17所述的方法，其特征在于，所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。The method of claim 17, wherein the template switching oligonucleotide contains three nucleotide residues at the 3'-end characterized by the molecular formula rGrG+N, where +N represents a locked core nucleotide residues.
  19. 如权利要求18所述的方法，其中所述模板转换寡核苷酸包含rGrG+G。The method of claim 18, wherein the template switching oligonucleotide comprises rGrG+G.
  20. 如权利要求10所述的方法，其中所述甲基供体是甜菜碱而所述金属盐是浓度至少为9mM的MgCl2。The method of claim 10, wherein said methyl donor is betaine and said metal salt is MgCl2 at a concentration of at least 9mM.
  21. 如权利要求9所述的方法，其中所述模板转换寡核苷酸选自由以下组成的组：The method of claim 9, wherein the template switching oligonucleotide is selected from the group consisting of:
    i.rGrG+G，优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，i.rGrG+G, preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
    ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N， ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
    iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and
    iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  22. 根据权利要求9所述的方法，其特征在于，所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。The method according to claim 9, characterized in that the cDNA is in a solution mixed with oligonucleotide primers or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by the polymerization of compound monomers.
  23. 根据权利要求22所述的方法，其中，所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNNNNNN，其中“N”是任何核苷碱基，而“V”是“A”或“C”或“G”。The method of claim 22, wherein the oligonucleotide primer comprises SEQ ID NO. 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N" is any nucleoside base, and "V" is "A" or "C" or "G".
  24. 一种分析多个单细胞中基因表达的方法，该方法包括以下步骤：根据权利要求9的方法制备cDNA文库；并对cDNA文库进行测序。A method for analyzing gene expression in multiple single cells, the method comprising the following steps: preparing a cDNA library according to the method of claim 9; and sequencing the cDNA library.
  25. 一种模板转换寡核苷酸(TSO)，其中其在3'-末端包含一个锁定的核苷酸残基。A template switching oligonucleotide (TSO) which contains a locked nucleotide residue at the 3'-end.
  26. 根据权利要求25所述的模板转换寡核苷酸，其中其在3'-末端包含三个核苷酸残基，所述核苷酸残基选自+N+N+N、N+N+N、NN+N、rN+N+N和rNrN+N，其中每次出现的N独立地是脱氧核糖核苷酸残基，每次出现的rN独立地是核糖核苷酸残基，并且每次出现的+N独立地是锁定的核苷酸残基。The template switching oligonucleotide according to claim 25, wherein it comprises three nucleotide residues at the 3'-end, and the nucleotide residues are selected from the group consisting of +N+N+N, N+N+ N, NN+N, rN+N+N, and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Each occurrence of +N is independently a locked nucleotide residue.
  27. 根据权利要求25的模板转换寡核苷酸，其中所述锁定核苷酸残基选自锁定的鸟嘌呤、锁定的腺嘌呤、锁定的尿嘧啶、锁定的胸腺嘧啶、锁定的胞嘧啶和锁定的5-甲基胞嘧啶。The template switching oligonucleotide according to claim 25, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  28. 根据权利要求27所述的模板转换寡核苷酸，其中所述三个核苷酸残基选自NN+G、rNrN+G、GG+N、rGrG+G和GG+G。The template switching oligonucleotide of claim 27, wherein the three nucleotide residues are selected from the group consisting of NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
  29. 根据权利要求9所述的方法，其特征在于，所述RNA是从选择性透过膜包围而 成的反应隔室中的单细胞中裂解释放出的RNA。The method of claim 9, wherein the RNA is surrounded by a selectively permeable membrane. RNA is released by cleavage of single cells in the reaction compartment.
  30. 根据权利要求29所述的单细胞，其特征在于，所述单细胞通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。The single cell according to claim 29, characterized in that the single cell is wrapped into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
  31. 根据权利要求9所述的方法，其中所述转座酶为Tn5转座酶。The method of claim 9, wherein the transposase is Tn5 transposase.
  32. 根据权利要求31所述的方法，其中所述转座子末端结构域包括Tn5转座子末端结构域。The method of claim 31, wherein the transposon terminal domain comprises a Tn5 transposon terminal domain.
  33. 根据权利要求9所述的方法，其中所述方法进一步包括将所述第一双链产物cDNA与第二双链产物DNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。The method of claim 9, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product DNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
  34. 根据权利要求9所述的方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。The method of claim 9, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
  35. 根据权利要求9所述的方法，其中所述的方法都在选择性透过膜包围而成的反应隔室中进行。The method of claim 9, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
  36. 一种在选择性透过膜包围而成的反应隔室中使用核糖核酸(RNA)样品制备二代测序(NGS)文库的方法，该方法包括：A method for preparing a next-generation sequencing (NGS) library using a ribonucleic acid (RNA) sample in a reaction compartment surrounded by a selectively permeable membrane, the method includes:
    (a)配制包括下述的组合物：RNA样品；第一链互补脱氧核糖核酸(cDNA)引物；模板开关寡核苷酸；逆转录酶；和dNTP；(a) Preparing a composition including: RNA sample; first-strand complementary deoxyribonucleic acid (cDNA) primer; template switch oligonucleotide; reverse transcriptase; and dNTP;
    将cDNA合成引物与RNA分子退火并合成第一条cDNA链以形成RNA-cDNA中间体；和通过使RNA-cDNA中间体与模板转换寡核苷酸(TSO)接触来进行逆转录酶反应，其中TSO在其3'-末端包含或不包含锁定核酸(LNA)，在适合于第一条DNA链延伸的条件下，与RNA分子互补，使其与TSO互补；annealing the cDNA synthesis primer to the RNA molecule and synthesizing the first cDNA strand to form an RNA-cDNA intermediate; and performing a reverse transcriptase reaction by contacting the RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein The TSO contains or does not contain a locked nucleic acid (LNA) at its 3'-end that is complementary to the RNA molecule under conditions suitable for extension of the first DNA strand, making it complementary to the TSO;
    (b)用包含转座酶和包含转座子末端结构域和第二标记后扩增引物结合结构域的转座子核酸的转座体标记产物mRNA/cDNA杂交双链以产生标记样品； (b) hybridizing double strands with a transposome labeled product mRNA/cDNA comprising a transposase and a transposon nucleic acid comprising a transposon terminal domain and a second post-labeling amplification primer binding domain to produce a labeled sample;
    (c)将所述加接头DNA片段进行PCR扩增，得到扩增产物，(c) PCR amplify the adapter-added DNA fragment to obtain an amplification product,
    RNA样品为权利要求1～5中任一项中涉及的所述待分析RNA与所述RNA捕获试剂连接成整体，或待分析RNA与所述RNA捕获试剂通过相互作用形成复合物，或待分析RNA与所述RNA捕获试剂经过生物或化学反应生成转化后形成的脱氧核糖核酸(DNA)和/或核糖核酸(RNA)的核酸内容物，The RNA sample is the RNA to be analyzed and the RNA capture reagent involved in any one of claims 1 to 5 connected as a whole, or the RNA to be analyzed and the RNA capture reagent form a complex through interaction, or the RNA to be analyzed is RNA and the RNA capture reagent undergo a biological or chemical reaction to generate the nucleic acid content of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) formed after conversion,
    其中(a)-(c)在权利要求1～6中任一项中涉及的反应隔室中进行。Wherein (a)-(c) are carried out in the reaction chamber according to any one of claims 1 to 6.
  37. 如权利要求36所述的方法，其中所述逆转录反应在甲基供体和金属盐存在下进行。The method of claim 36, wherein the reverse transcription reaction is performed in the presence of a methyl donor and a metal salt.
  38. 如权利要求37所述的方法，其特征在于，所述甲基供体是甜菜碱。The method of claim 37, wherein the methyl donor is betaine.
  39. 如权利要求37所述的方法，其特征在于，所述金属盐是镁盐。The method of claim 37, wherein the metal salt is a magnesium salt.
  40. 如权利要求37所述的方法，其中所述镁盐具有至少7mM的浓度。The method of claim 37, wherein the magnesium salt has a concentration of at least 7mM.
  41. 如权利要求35所述的方法，其中所述模板转换寡核苷酸包含一个或两个核糖核苷酸残基和所述0个或一个以上锁核酸(LNA)残基。The method of claim 35, wherein said template switching oligonucleotide comprises one or two ribonucleotide residues and said zero or more locked nucleic acid (LNA) residues.
  42. 如权利要求41所述的方法，其中所述一个或两个核糖核苷酸残基是核糖鸟嘌呤。The method of claim 41, wherein said one or two ribonucleotide residues are riboguanine.
  43. 如权利要求41所述的方法，其中所述锁核酸残基选自锁鸟嘌呤、锁腺嘌呤、锁尿嘧啶、锁胸腺嘧啶、锁胞嘧啶和锁5-甲基胞嘧啶。The method of claim 41, wherein the locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  44. 如权利要求43所述的方法，其特征在于，所述锁核酸残基是锁鸟嘌呤。The method of claim 43, wherein the locked nucleic acid residue is locked guanine.
  45. 如权利要求36所述的方法，其特征在于，所述模板转换寡核苷酸在3'-末端包含三个以分子式rGrG+N为特征的核苷酸残基，其中+N代表锁定的核苷酸残基。The method of claim 36, wherein the template switching oligonucleotide contains three nucleotide residues at the 3'-end characterized by the molecular formula rGrG+N, where +N represents a locked core nucleotide residues.
  46. 如权利要求45所述的方法，其中所述模板转换寡核苷酸包含rGrG+G。 The method of claim 45, wherein the template switching oligonucleotide comprises rGrG+G.
  47. 根据权利要求37所述的方法，其中所述甲基供体是甜菜碱，而所述金属盐是浓度至少为9mM的MgCl2。The method of claim 37, wherein said methyl donor is betaine and said metal salt is MgCl2 at a concentration of at least 9mM.
  48. 如权利要求36所述的方法，其中所述模板转换寡核苷酸选自由以下组成的组：The method of claim 36, wherein the template switching oligonucleotide is selected from the group consisting of:
    i.rGrG+G，优选为SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G，i.rGrG+G, preferably SEQ ID NO.1:AAGCAGTGGTATCAACGCAGAGTACrGrG+G,
    ii.rGrG+N，优选为SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N，ii.rGrG+N, preferably SEQ ID NO.2:AAGCAGTGGTATCAACGCAGAGTACrGrG+N,
    iii.+G+G+G，优选SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G和iii.+G+G+G, preferably SEQ ID NO.3:AAGCAGTGGTATCAACGCAGAGTAC+G+G+G and
    iv.rG+G+G，优选为SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G。iv.rG+G+G, preferably SEQ ID NO.4:AAGCAGTGGTATCAACGCAGAGTACrG+G+G.
  49. 根据权利要求37所述的方法，其特征在于，所述cDNA是在混有寡核苷酸引物的溶液中或者在由带双键的寡核苷酸引物与一种或多种带双键的化合物单体聚合而成的胶状高分子化合物上合成的。The method of claim 37, wherein the cDNA is in a solution mixed with oligonucleotide primers or in a solution composed of an oligonucleotide primer with a double bond and one or more oligonucleotide primers with a double bond. It is synthesized from a colloidal polymer compound formed by the polymerization of compound monomers.
  50. 根据权利要求49所述的方法，其中所述寡核苷酸引物包含SEQ ID NO.5:5’-AAGCAGTGGTATCAACGCAGAGTACT30VN和/或NNNNNNNNNN，其中“N”是任何核苷碱基，而“V”是“A”或“C”或“G”。The method of claim 49, wherein the oligonucleotide primer comprises SEQ ID NO.5: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN and/or NNNNNNNNNN, wherein "N" is any nucleoside base, and "V" is " A" or "C" or "G".
  51. 一种分析多个单细胞中基因表达的方法，该方法包括以下步骤：使用根据权利要求36的方法制备cDNA文库；并对cDNA文库进行测序。A method for analyzing gene expression in multiple single cells, the method comprising the steps of: preparing a cDNA library using the method according to claim 36; and sequencing the cDNA library.
  52. 一种模板转换寡核苷酸(TSO)，在其3'-最末端包含一个锁定的核苷酸残基。A template-switching oligonucleotide (TSO) containing a locked nucleotide residue at its 3'-most end.
  53. 根据权利要求52所述的模板转换寡核苷酸，其在3'-末端包含三个核苷酸残基，所述核苷酸残基选自+N+N+N、N+N+N、NN+N、rN+N+N和rNrN+N，其中每次出现的N独立地是脱氧核糖核苷酸残基，每次出现的rN独立地是核糖核苷酸残基，并且每次出现的+N独立地是锁定的核苷酸残基。The template switching oligonucleotide according to claim 52, comprising three nucleotide residues at the 3'-end, the nucleotide residues being selected from the group consisting of +N+N+N, N+N+N , NN+N, rN+N+N and rNrN+N, where each occurrence of N is independently a deoxyribonucleotide residue, each occurrence of rN is independently a ribonucleotide residue, and each occurrence of Occurrences of +N are independently locked nucleotide residues.
  54. 权利要求52的模板转换寡核苷酸，其中所述锁定核苷酸残基选自锁定的鸟嘌呤、 锁定的腺嘌呤、锁定的尿嘧啶、锁定的胸腺嘧啶、锁定的胞嘧啶和锁定的5-甲基胞嘧啶。The template switching oligonucleotide of claim 52, wherein the locked nucleotide residue is selected from the group consisting of locked guanine, Locked adenine, locked uracil, locked thymine, locked cytosine and locked 5-methylcytosine.
  55. 如权利要求53所述的模板转换寡核苷酸，其中所述三个核苷酸残基选自NN+G、rNrN+G、GG+N、rGrG+G和GG+G。The template switching oligonucleotide of claim 53, wherein the three nucleotide residues are selected from the group consisting of NN+G, rNrN+G, GG+N, rGrG+G and GG+G.
  56. 如权利要求54所述的方法，其特征在于，所述RNA是选择性透过膜包围而成的反应隔室中的单细胞中裂解释放出的RNA。The method of claim 54, wherein the RNA is RNA released by cleavage of single cells in a reaction compartment surrounded by a selectively permeable membrane.
  57. 根据权利要求56所述的单细胞，其特征在于，通过微流控系统包裹进选择性透过膜包围而成的反应隔室中。The single cell according to claim 56, characterized in that it is wrapped into a reaction compartment surrounded by a selectively permeable membrane through a microfluidic system.
  58. 根据权利要求36所述的方法，其中所述转座酶包括Tn5转座酶。The method of claim 36, wherein said transposase comprises Tn5 transposase.
  59. 根据权利要求58所述的方法，其中所述转座子末端结构域包括Tn5转座子末端结构域。The method of claim 58, wherein the transposon terminal domain comprises a Tn5 transposon terminal domain.
  60. 根据权利要求36所述的方法，其中所述方法进一步包括将第一双链产物cDNA与第二双链产物cDNA合并以产生合并的cDNA样品，然后标记所述合并的cDNA样品。The method of claim 36, wherein the method further comprises combining the first double-stranded product cDNA with the second double-stranded product cDNA to produce a pooled cDNA sample, and then labeling the pooled cDNA sample.
  61. 根据权利要求36所述的方法，其中所述方法还包括对所述RNA样品的一种或多种RNA种类进行定量。The method of claim 36, wherein the method further comprises quantifying one or more RNA species of the RNA sample.
  62. 根据权利要求36所述的方法，其中所述方法都在选择性透过膜包围而成的反应隔室中进行。 The method of claim 36, wherein the method is performed in a reaction compartment surrounded by a selectively permeable membrane.
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