Method for synthesizing double-stranded cdna and use thereof

Abstract

Provided are a method for synthesizing double-stranded cDNA and the use thereof. The method comprises: performing a reverse transcription reaction on an mRNA template by means of using a reverse transcription primer and a reverse transcription mixture, so as to obtain double-stranded cDNA, wherein the reverse transcription mixture comprises a reverse transcriptase having a template switching function, a template switch oligo and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switch oligo is 2-12.5 μM, and the final concentration of dNTPs is 1-10 mM. The reverse transcription reaction is performed by means of using the reverse transcription mixture. By means of optimizing the amounts of the dNTPs, the template switch oligo and the reverse transcriptase involved in the reaction in the reverse transcription reaction system, the efficiency of the reverse transcription reaction is improved, enabling the production of a double-stranded cDNA product in a one-step reaction, thereby greatly shortening the reverse transcription reaction time and thus improving the quality of the double-stranded cDNA product.

Description

Method for synthesizing double-stranded cDNA and its application Technical Field

[0001] The present invention relates to the technical field of gene synthesis, and in particular to a method for synthesizing double-stranded cDNA and its application. Background Art

[0002] Conventional double-stranded cDNA synthesis in the prior art is primarily accomplished through the following process: First, mRNA or RNA is reverse-transcribed into a single cDNA strand using a poly-T primer or random primers N6 / N8, with the help of reverse transcriptase and dNTPs; the reverse transcription reaction takes 15 minutes to 1 hour. RNase H and DNA polymerase are then added (or, alternatively, DNA polymerase is omitted, utilizing the DNA polymerase function of the reverse transcriptase in the system). RNase H nicks the RNA in the DNA / RNA hybrid, and DNA polymerase then uses the nicked RNA strand as a primer to synthesize the double-stranded cDNA. The resulting double-stranded cDNA is not complete DNA and contains a small amount of RNA sequence.

[0003] The reverse transcription process in existing spatiotemporal transcriptome construction also requires a two-step reaction to synthesize double-stranded cDNA. Furthermore, the synthesis of the second strand requires the addition of additional reaction buffer and enzymes, resulting in low reaction efficiency and low quality of the resulting spatiotemporal transcriptome. The existing spatiotemporal transcriptome construction method is as follows: the 3'-end poly A of the mRNA is bound to the poly T position on the spatiotemporal chip for reverse transcription to synthesize the first strand of cDNA. The reverse transcriptase transcribes three additional C bases at the 3' end of the first strand of cDNA. A template switch oligo (TSO) containing three G bases is added to switch the template. The reverse transcriptase then uses this primer as a template to add a TSO sequence to the 3' end of the first strand of cDNA. Double-stranded cDNA double-strand synthesis reagents, including DNA polymerase and double-stranded synthesis primers, are added to the chip used for first-strand cDNA synthesis. The synthesized double-stranded cDNA is dissociated from the chip by alkaline lysis, and the alkali is neutralized with Tris-HCl. PCR amplification is performed using the sequences of adapter 1 and TSO as primers to obtain double-stranded cDNA; the double-stranded cDNA is broken and then added to adapter 2; PCR amplification is performed using primers of adapter 1 and adapter 2 to obtain a library containing position tags and mRNA sequences, which can be sequenced and analyzed.

[0004] Therefore, how to provide a cDNA synthesis method that is simple to operate and has fewer impurities is very important for the application and development of related technologies involving reverse transcription.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for synthesizing double-stranded cDNA and its application, so as to solve the problems of low efficiency and multiple steps in the prior art of double-stranded cDNA synthesis.

[0007] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for synthesizing a double-stranded cDNA is provided, the method comprising: performing a reverse transcription reaction on an mRNA template using a reverse transcription primer and a reverse transcription mixture to obtain a double-stranded cDNA; wherein the reverse transcription mixture comprises a reverse transcriptase having a template switching function, a template switching primer, and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

[0008] Furthermore, the final concentration of reverse transcriptase is 8-12 U / μL, the final concentration of template switching primer is 2.5-5 μM, and the final concentration of dNTPs is 4-6 mM; preferably, the final concentration of reverse transcription primer is 0.5-5 μM; preferably, the reverse transcription mixture also includes: reverse transcription buffer and RNase inhibitor.

[0009] Furthermore, the reverse transcription reaction includes: synthesizing the first-chain cDNA from the mRNA template using a reverse transcription primer and a reverse transcriptase to obtain a first-chain cDNA product bound to a template-switching primer; and reversely synthesizing the second-chain cDNA from the first-chain cDNA product using a template-switching primer and a reverse transcriptase to obtain a double-stranded cDNA. Preferably, the reaction temperatures for the first-chain cDNA synthesis and the second-chain cDNA synthesis are each independently 42-50°C.

[0010] Furthermore, the template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; preferably, the length of the template switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0011] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a method for constructing a spatiotemporal transcriptome library is provided, which comprises: combining an mRNA template with a reverse transcription primer fixed on a spatiotemporal chip, adding a reverse transcription mixture, performing a reverse transcription reaction, and obtaining a cDNA double strand; dissociating the cDNA double strand to obtain a free cDNA second strand; performing a PCR reaction using the free cDNA second strand as a template to obtain a free cDNA double strand; constructing a fragmentation library of the free cDNA double strand to obtain a spatiotemporal transcriptome library; wherein the reverse transcription mixture comprises a reverse transcriptase with a template switching function, a template switching primer, and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

[0012] Furthermore, the final concentration of reverse transcriptase is 8-12 U / μL, the final concentration of template switching primer is 2.5-5 μM, and the final concentration of dNTPs is 4-6 mM; preferably, the final concentration of reverse transcription primer is 0.5-5 μM; preferably, the reverse transcription mixture also includes: reverse transcription buffer and RNase inhibitor.

[0013] Furthermore, the reverse transcription reaction includes: synthesizing the first-chain cDNA from the mRNA template using a reverse transcription primer and a reverse transcriptase to obtain a first-chain cDNA product bound to a template-switching primer; and reversely synthesizing the second-chain cDNA from the first-chain cDNA product using a template-switching primer and a reverse transcriptase to obtain a double-stranded cDNA. Preferably, the dissociation includes alkaline lysis of the double-stranded cDNA and neutralization of the resulting lysate, wherein the lysate includes a free second-chain cDNA. Preferably, the reaction temperatures for the first-chain cDNA synthesis and the second-chain cDNA synthesis are each independently 42-50°C.

[0014] Furthermore, the template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; preferably, the length of the template switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0015] To achieve the above object, according to a third aspect of the present invention, a kit is provided, which comprises a reverse transcriptase with a template switching function, a template switching primer, a reverse transcription primer and dNTPs; wherein the working concentration of the reverse transcriptase is 5-20 U / μL, the working concentration of the template switching primer is 2-12.5 μM, and the working concentration of the dNTPs is 1-10 mM; preferably, the working concentration of the reverse transcriptase is 8-12 U / μL, the working concentration of the template switching primer is 2.5-5 μM, and the working concentration of the dNTPs is 4-6 mM ; Preferably, the working concentration of the reverse transcription primer is 0.5-5 μM; preferably, the kit further comprises: a reverse transcription buffer and an RNase inhibitor; preferably, the template switching primer comprises: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; preferably, the length of the template switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0016] To achieve the above object, according to the fourth aspect of the present invention, a RT-qPCR detection method is provided, which comprises: reverse transcribing the RNA to be tested using the above method to obtain a cDNA double strand; and performing a fluorescent PCR reaction using the cDNA double strand as a template.

[0017] In order to achieve the above object, according to the fifth aspect of the present invention, a method for constructing a transcriptome library is provided, which comprises: reverse transcribing the test RNA using the above method to obtain a cDNA double strand; and constructing a fragmentation library of the cDNA double strand to obtain a transcriptome library.

[0018] Furthermore, the transcriptome library includes a single-cell transcriptome library.

[0019] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, a sequencing method is provided, which comprises: sequencing the spatiotemporal transcriptome library constructed by the above-mentioned method for constructing a spatiotemporal transcriptome library, or the transcriptome library obtained by the above-mentioned method, to obtain sequencing results.

[0020] In order to achieve the above object, according to the seventh aspect of the present invention, there is provided a use of the above kit in cDNA double-strand synthesis.

[0021] By applying the technical solution of the present invention, during the process of cDNA synthesis, the reverse transcription mixture of the present application is used to carry out the reverse transcription reaction. By optimizing the amounts of dNTPs, template switching primers and reverse transcriptase involved in the reverse transcription reaction system, the efficiency of the reverse transcription reaction is improved, and a cDNA double-stranded product can be obtained in a one-step reaction, which greatly shortens the reaction time of reverse transcription and improves the quality of the product cDNA double-strand. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] FIG1 shows a schematic diagram of the process of constructing a spatiotemporal transcriptome in the prior art;

[0024] FIG2 shows a schematic diagram of the principle of double-stranded cDNA synthesis of the present application;

[0025] FIG3 shows a 2100 quality control graph of the cDNA fragment synthesized using the technical solution of the present application in Example 1 of the present application;

[0026] FIG4 shows a 2100 quality control graph of the cDNA fragment obtained using the prior art solution in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0027] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] Explanation of terms:

[0029] First strand of cDNA: The first cDNA strand obtained by reverse transcription of mRNA, also referred to as the first strand of cDNA in this application.

[0030] cDNA second strand: The strand obtained by further PCR amplification using the first cDNA strand obtained by reverse transcription of mRNA as a template, also referred to as cDNA second strand in this application.

[0031] As mentioned in the background art, existing double-stranded cDNA synthesis requires a two-step reaction to complete, and when synthesizing the second-strand cDNA, a reaction buffer and related enzymes need to be added to the system before the reaction. It can be seen that the existing technology for synthesizing cDNA has low synthesis efficiency and the risk of contamination, and the resulting double-stranded cDNA may contain some impurities. Therefore, the present application intends to provide a method for efficiently synthesizing double-stranded cDNA.

[0032] In a first typical embodiment of the present application, a method for synthesizing a double-stranded cDNA is provided, the method comprising: reverse transcribing an mRNA template using a reverse transcription primer and a reverse transcription mixture to obtain a double-stranded cDNA; wherein the reverse transcription mixture comprises a reverse transcriptase with a template switching function, a template switching primer and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

[0033] Wherein, the reverse transcriptase of the present application has the DNA polymerase activity of reverse transcription, template switching, strand displacement and DNA dependence, can complete reverse transcription, mRNA template displacement and the whole process of cDNA double-strand synthesis with one cDNA chain as template.And because the reverse transcription mixed solution containing this enzyme is added once before all reactions, in order to control the degree of more complete reaction of each reaction in the cDNA double-strand synthesis process, it is necessary to carry out more accurate dosage control to each component in the reverse transcription mixed solution.Through various test screening processes, the reverse transcription mixed solution with the above-mentioned components and relevant concentrations of the present application is obtained.Can in the same reaction system, only one step is generated to obtain cDNA double-strand, without the need to add any reaction component when the second-chain cDNA is synthesized, thereby greatly improving the synthesis efficiency of the cDNA double-strand, and the quality of the cDNA double-strand obtained in a relatively short time is also high, which can provide certain quality assurance for other technologies related to cDNA double-strand synthesis.

[0034] To further stabilize the cDNA reaction environment and improve cDNA synthesis efficiency, in a preferred embodiment, the final concentration of reverse transcriptase is 8-12 U / μL, the final concentration of the template-switching primer is 2.5-5 μM, the final concentration of dNTPs is 4-6 mM, and the final concentration of the reverse transcription primer is 0.5-5 μM. To further enhance enzyme activity and provide an enzyme reaction environment with an appropriate pH value, in a preferred embodiment, the reverse transcription mixture also includes a reverse transcription buffer and an RNase inhibitor; the reverse transcription buffer includes Tris-HCl, 75-100 mM KCl, 3-5 mM MgCl2, and 2.5-5 mM DTT at a final concentration of 50-100 mM in the reverse transcription buffer. Furthermore, to further stabilize the activity of the cDNA synthesis product, in a preferred embodiment, the reverse transcription mixture also includes betaine, trehalose, Triton X-100, NP40, and NaCl.

[0035] The present application only requires one step for double-stranded cDNA synthesis, and there is no need to add additional reaction components to the reaction system after the synthesis of one chain of cDNA. In a preferred embodiment, as shown in Figure 2, the reverse transcription reaction includes: using a reverse transcription primer and a reverse transcriptase to synthesize the first chain of cDNA on the mRNA template to obtain a first chain of cDNA product bound to a template conversion primer; using a template conversion primer and a reverse transcriptase to reversely synthesize the second chain of cDNA on the first chain of cDNA product to obtain a double-stranded cDNA. After the present application reversely transcribes to obtain one chain of cDNA, there is no need to additionally add DNA polymerase and double-stranded synthesis primer to complete the replacement of the double-stranded cDNA synthesis template. In order to further improve the efficiency of cDNA synthesis, in a preferred embodiment, the reaction temperature of cDNA synthesis is 42-50°C, and the reaction time is 2-3h.

[0036] Since the reverse transcriptase used in the present application has the characteristics of template switching and strand displacement, it generates an additional end at the 3' end of the cDNA that can be complementary to the template switching primer when performing single-strand cDNA synthesis. In a preferred embodiment, the template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; to improve the accuracy and efficiency of primer complementarity, in a preferred embodiment, the length of the template switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0037] Among them, locked nucleic acid (LNA) is a modified RNA and a synthetic nucleic acid analog containing a bridged bicyclic sugar moiety. The methylene group added between the 2'-O- and 4'-C positions "locks" the ribofuranose ring in a 3'-endo conformation. Due to the constraints of this bicyclic sugar backbone, LNA can only form A-type duplexes. In addition, LNA fully adheres to the Watson-Crick base pairing principle. Therefore, LNA:DNA hybrid duplexes can spontaneously form from sequence-complementary DNA and LNA, and studies have found that the annealing temperature of LNA:DNA hybrids is significantly higher than that of their DNA:DNA counterparts.

[0038] In a second typical embodiment of the present application, a method for constructing a spatiotemporal transcriptome library is provided, the method comprising: combining an mRNA template with a reverse transcription primer fixed on a spatiotemporal chip, adding a reverse transcription mixture, performing a reverse transcription reaction, and obtaining a cDNA double strand; dissociating the cDNA double strand to obtain a free cDNA second strand; performing a PCR reaction using the free cDNA second strand as a template to obtain a free cDNA double strand; constructing a cDNA library by fragmenting the free cDNA double strand to obtain a spatiotemporal transcriptome library; wherein the reverse transcription mixture comprises a reverse transcriptase with a template switching function, a template switching primer, and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

[0039] Spatiotemporal transcriptomics is a technology that studies the gene expression of organisms at different time points and spatial locations. The process of reverse transcription and cDNA double-strand synthesis for library construction is related to the accuracy of subsequent spatiotemporal transcriptome analysis. Using the method of constructing spatiotemporal transcriptomics in this application, higher quality transcriptome samples can be obtained efficiently.

[0040] Wherein, the reverse transcriptase of the present application has the DNA polymerase activity of reverse transcription, template switching, strand displacement and DNA dependence, can complete reverse transcription, mRNA template displacement and the whole process of cDNA double-strand synthesis with one chain of cDNA as template.And because the reverse transcription mixed solution containing this enzyme is added once before all reactions, in order to control the degree of more complete reaction of each reaction in the cDNA double-strand synthesis process, it is necessary to carry out more accurate dosage control to each component in the reverse transcription mixed solution.Through various test screening processes, obtain the reverse transcription mixed solution with the above-mentioned composition and relevant concentration of the present application.Can in the same reaction system, only one step is generated to obtain cDNA double-strand, need not add any reaction component extra again when second-chain cDNA is synthesized, and then greatly improve the synthesis efficiency of cDNA double-strand, the quality of the cDNA double-strand obtained in a relatively short time is also high, can provide certain quality assurance for spatiotemporal transcriptome analysis.

[0041] To further stabilize the cDNA reaction environment and improve cDNA synthesis efficiency, in a preferred embodiment, the final concentration of reverse transcriptase is 8-12 U / μL, the final concentration of the template-switching primer is 2.5-5 μM, the final concentration of dNTPs is 4-6 mM, and the final concentration of the reverse transcription primer is 0.5-5 μM. To enhance enzyme activity and provide an appropriate pH for the enzyme reaction, in a preferred embodiment, the reverse transcription mixture also includes a reverse transcription buffer and an RNase inhibitor. The reverse transcription buffer includes Tris-HCl, 75-100 mM KCl, 3-5 mM MgCl2, and 2.5-5 mM DTT at a final concentration of 50-100 mM in the reverse transcription buffer. Furthermore, to stabilize the activity of the cDNA synthesis product, in a preferred embodiment, the reverse transcription mixture also includes betaine, trehalose, Triton X-100, NP40, and NaCl.

[0042] In the process of constructing the spatiotemporal transcriptome of the present application, the synthesis of double-stranded cDNA only requires one step, and there is no need to add additional reaction components to the reaction system after the synthesis of one chain of cDNA. In a preferred embodiment, the reverse transcription reaction includes: using a reverse transcription primer and a reverse transcriptase to synthesize the first chain of cDNA from the mRNA template to obtain a first chain of cDNA product bound to a template conversion primer; using a template conversion primer and a reverse transcriptase to reversely synthesize the second chain of cDNA from the first chain of cDNA product to obtain a double-stranded cDNA. After the present application reversely transcribes the first chain of cDNA, there is no need to add additional DNA polymerase and double-stranded synthesis primer to complete the replacement of the double-stranded cDNA synthesis template. In order to further improve the efficiency of cDNA synthesis, in a preferred embodiment, the reaction temperature of cDNA synthesis is 42-50°C, and the reaction time is 2-3h.

[0043] During the spatiotemporal transcriptome construction process of this application, double-stranded cDNA is synthesized on the chip, and the second-stranded cDNA needs to be dissociated from the chip for further library construction. In a preferred embodiment, the dissociation includes alkaline lysis of the double-stranded cDNA and neutralization of the resulting lysate, which includes the free second-stranded cDNA. In a preferred embodiment, the alkaline lysis includes potassium hydroxide at a final concentration of 0.08M-0.4M, and the neutralization includes Tris-HCl.

[0044] Since the reverse transcriptase used in the present application has the characteristics of template switching and strand displacement, it generates an additional end at the 3' end of the cDNA that can be complementary to the template switching primer when performing single-strand cDNA synthesis. In a preferred embodiment, the template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; to improve the accuracy and efficiency of primer complementarity, in a preferred embodiment, the length of the template switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0045] In a third typical embodiment of the present application, a kit is provided, which includes a reverse transcriptase with template switching function, a template switching primer, a reverse transcription primer and dNTPs; the working concentration of the reverse transcriptase is 5-20 U / μL, the working concentration of the template switching primer is 2-12.5 μM, and the working concentration of the dNTPs is 1-10 mM; in a preferred embodiment, the working concentration of the reverse transcriptase is 8-12 U / μL, the working concentration of the template switching primer is 2.5-5 μM, and the working concentration of the dNTPs is 4-6 mM; the reverse transcriptase in this kit has reverse transcription, template switching, chain displacement and DNA-dependent DNA polymerase activities, and can complete the entire process of reverse transcription, mRNA template displacement and double-stranded cDNA synthesis using one cDNA chain as a template. The reverse transcription mixture containing the above-mentioned components and relevant concentrations can generate double-stranded cDNA in only one step in the same reaction system, without the need to add any additional reaction components during the synthesis of the double-stranded cDNA, thereby greatly improving the synthesis efficiency of the double-stranded cDNA and more conveniently obtaining high-quality double-stranded cDNA products.

[0046] In order to further stabilize the cDNA reaction environment and improve the efficiency of cDNA synthesis, in a preferred embodiment, the kit further comprises: a reverse transcription buffer and an RNase inhibitor; preferably, the working concentration of the reverse transcription primer is 0.5-5 μM; preferably, the reverse transcription buffer comprises Tris-HCl, 75-100 mM KCl, 3-5 mM MgCl2 and 2.5-5 mM DTT at a final concentration of 50-100 mM in the reverse transcription buffer; preferably, the kit further comprises: betaine, trehalose, Triton X-100, NP40 and NaCl. In a preferred embodiment, the template-switching primer includes: a template-switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; to improve the accuracy and efficiency of primer complementarity, in a preferred embodiment, the length of the template-switching primer is 20-35 nt; preferably, the mRNA template is derived from tissues or cells.

[0047] In a fourth typical embodiment of the present application, an RT-qPCR detection method is provided, which includes: reverse transcribing the RNA to be tested using the above method to obtain a cDNA double strand; and performing a fluorescent PCR reaction using the cDNA double strand as a template.

[0048] In a fifth typical embodiment of the present application, a method for constructing a transcriptome library is provided, which comprises: reverse transcribing the test RNA using the above-mentioned method to obtain a cDNA double strand; constructing a fragmentation library of the cDNA double strand to obtain a transcriptome library.

[0049] In a preferred embodiment, the transcriptome library comprises a single-cell transcriptome library, and preferably, the RNA to be detected is derived from a single-cell sample.

[0050] The double-stranded cDNA synthesis method of the present application is applicable to the construction of any RNA library. Depending on the fragment size of the sample, it can be selected whether to fragment the cDNA, including but not limited to physical shearing or chemical enzymatic shearing methods; and depending on the type of connector used to construct the library, it can be selected whether to end-repair the cDNA fragments before connecting the cDNA sequencing connector.

[0051] In a sixth typical embodiment of the present application, a sequencing method is provided, comprising: sequencing the spatiotemporal transcriptome library constructed by the above-mentioned method for constructing a spatiotemporal transcriptome library, or the transcriptome library obtained by the above-mentioned method, to obtain sequencing results.

[0052] In a seventh typical embodiment of the present application, a method for using the above-mentioned kit in double-stranded cDNA synthesis is provided.

[0053] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0054] Example 1 (Construction of spatiotemporal transcriptome using the method of this application (as shown in FIG2 ))

[0055] In the following process, the chip, RT oligo, and permeabilization reagent were from the Stereo-seq Transcriptome Reagent Set (Cat. No.: 201ST114), the fragmentation and PCR reagents were from the Stereo-seq Library Construction Kit (Cat. No.: 101KL114), the RT buffer and RT enzyme were purchased from BGI (Cat. No.: LS-EZ-E-00027Q), the RNase inhibitor was purchased from BGI (Cat. No.: 1000014382), and 25 mM dNTP was purchased from Shanghai Bioengineering (Cat. No.: B110045-0005).

[0056] The tissue used for constructing the spatiotemporal transcriptome was mouse brain tissue.

[0057] 1. Tissue patching and fixation

[0058] 1.1 Remove the Stereo-seq chip T carrier from the vacuum-dried aluminum foil bag and record the number on the back of the chip;

[0059] Be careful not to touch the front of the chip. Allow to warm to room temperature for 1 minute.

[0060] 1.2 Adjust the PCR instrument temperature to 37°C and the heated cover temperature to 42°C in advance, and place the PCR adapter to balance the temperature.

[0061] 1.3 Pre-cool methanol: Add enough methanol to a slide box or 50 mL centrifuge tube to ensure that the methanol is sufficient to immerse all chips (you can place a regular slide in the container to check whether the volume of methanol is sufficient). Cover the container and pre-cool the methanol at -20°C for 5-30 minutes.

[0062] 1.4 Cut 10 μm tissue slices on a freezing microtome and flatten them. Align the front of the chip with the tissue for mounting.

[0063] 1.5 Quickly place the patched vector on the PCR adapter and incubate at 37°C for 5 minutes to dry.

[0064] 1.6 Immediately place the dried chips in -20°C pre-cooled methanol for 30 minutes to fix them, ensuring that all chips are immersed in methanol.

[0065] 1.7 After fixation, transfer the slide box or 50 mL centrifuge tube to a fume hood;

[0066] 1.8 Remove the carrier from the slide box or 50mL centrifuge tube and use dust-free paper to absorb excess methanol on the back and around the slide to ensure that there is no liquid residue in the gaps between the chips;

[0067] 1.9 Place the carrier upright on a slide staining rack and ventilate in a fume hood for 4-6 minutes to allow the methanol to evaporate completely;

[0068] 1.10 After the methanol evaporates and the tissue turns white, transfer the carrier to the lab table. Assemble the gasket and fixture to form a carrier, and secure the chip to the carrier to form a handheld carrier.

[0069] 2. Tissue Permeabilization

[0070] 2.1 Prepare 1× Permeabilization Reagent Working Solution by preparing 2 mL of 0.01 N HCl in advance. Dissolve PR Enzyme (red cap, powdered) in 1 mL of freshly prepared 0.01 N HCl and mix thoroughly by pipetting. Then, dilute 10 μL of the 10× Permeabilization Reagent Stock Solution to 100 μL with 0.01 N HCl. Label this solution as 1× Permeabilization Reagent Working Solution.

[0071] 2.2 Set the temperature of two PCR machines (or one PCR machine and one metal bath) to 37°C and the heated cover temperature to 42°C in advance. Place a PCR adapter in one of the PCR machines to balance the temperature.

[0072] 2.3 Place the handheld carrier on the PCR adapter, cover the PCR instrument, and rewarm at 37°C for 3 minutes. At the same time, place the 1× permeabilization reagent working solution in the PCR instrument or metal bath and rewarm at 37°C for 3 minutes.

[0073] 2.4 After rewarming, add 1× permeabilization reagent working solution to one corner of the chip at a volume of 100 μL / chip. Seal the handheld carrier with sealing film and cover the PCR instrument.

[0074] 2.5 Incubate the permeabilization reaction at 37°C for 11 minutes.

[0075] 3. Reverse transcription (reverse transcription process of this application)

[0076] 3.1 Thaw RT buffer, 25mM dNTP, and RT oligo in advance and place on ice;

[0077] 3.2 While waiting for permeabilization, prepare the RT reaction mixture according to Table 1 and place it on ice;

[0078] Table 1 RT reaction mixture

[0079] 3.3 During permeabilization, adjust the reaction temperature of another PCR instrument to 42°C and the heated lid temperature to 47°C. Place another PCR adapter to balance the temperature.

[0080] 3.4 Remove the permeabilized handheld carrier from the PCR instrument;

[0081] 3.5 Slightly tilt the handheld carrier to an angle of less than 20° and use a pipette to remove the permeabilization reagent from one corner of the chip;

[0082] Note: When tearing off the sealing film, do not press the upper part of the clamp buckle on both sides to prevent the carrier from falling off.

[0083] 3.6 Add 100 μL of PR Rinse Buffer solution;

[0084] 3.7 Slightly tilt the handheld carrier to an angle of less than 20° and use a pipette to aspirate the PR Rinse Buffer solution at one corner of the chip to keep the chip moist.

[0085] NOTE: Avoid allowing the chip to dry out completely.

[0086] 3.8 Take out the prepared RT reaction mixture, pipette and mix thoroughly, then centrifuge briefly. Add the RT reaction mixture to one corner of the chip to ensure that the RT reaction mixture evenly covers the entire chip. The chip is loaded with a reverse transcription primer consisting of TTGTCTTCCTAAGACNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTV as shown in SEQ ID NO: 1, where N is any one of ATCG and V is any of three nucleotides other than T.

[0087] 3.9 Seal the handheld carrier with a plate sealer, place it on the PCR adapter of a 45°C PCR instrument, close the PCR instrument lid, and incubate for 2 hours.

[0088] 4. Secondary chain elution

[0089] 4.1 Adjust the reaction temperature of a PCR instrument to 70°C and the hot cover temperature to 75°C in advance, and place a PCR adapter to balance the temperature.

[0090] 4.2 Dilute 8M KOH to 0.1M with enzyme-free water, vortex to mix evenly, centrifuge briefly, and set aside;

[0091] 4.3 After the reverse transcription reaction is completed, remove the handheld carrier from the PCR instrument, tilt the handheld carrier slightly to an angle of less than 20°, and use a pipette to aspirate the reverse transcription reaction solution at one corner of the chip;

[0092] 4.4 Add 100 μL of 0.1 M KOH to the chip, ensuring that the KOH evenly covers the entire chip;

[0093] 4.5 Seal the handheld carrier with a sealing film, place it on the PCR adapter of a 70°C PCR instrument, close the PCR instrument lid, and react for 20 minutes;

[0094] 4.6 Remove the handheld carrier from the PCR instrument after the reaction is completed. Slightly tilt the handheld carrier at an angle of less than 20°. Use a pipette to aspirate the KOH solution from one corner of the chip and transfer it to a 1.5 mL tube. Then add 4 μL of 1 M Tris-HCl (pH 6.8) and mix thoroughly with a pipette. Then divide the solution into two PCR tubes for separate PCR reactions.

[0095] 5.PCR and purification

[0096] 5.1 Prepare the PCR Mix according to Table 2;

[0097] Table 2 PCR Mix

[0098] 5.2 Vortex to mix, centrifuge briefly, and place in a PCR instrument. Amplify according to the reaction program in Table 3 below.

[0099] Table 3 PCR amplification program

[0100] 5.3 Mix the above PCR product and magnetic beads equilibrated at room temperature at a ratio of 1:0.6 (120 μL PCR product, 72 μL magnetic beads), shake well, and incubate at room temperature for 10 minutes;

[0101] 5.4 After instant centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3-5 minutes until the liquid becomes clear. Use a pipette to carefully remove the supernatant and discard it.

[0102] 5.5 Keep the centrifuge tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol. Rinse the magnetic beads by rotating the centrifuge tube on the magnetic stand. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0103] 5.6 Repeat step 5.5 once;

[0104] 5.7 Try to drain the liquid in the tube. If a small amount of liquid remains on the tube wall, centrifuge the tube instantly, separate it on a magnetic stand, and then use a small-scale pipette to drain the liquid at the bottom of the tube.

[0105] 5.8 Let the beads air-dry at room temperature for 3-5 minutes until the surface of the beads is free of reflections and cracks.

[0106] 5.9 Add 100 μL of TE buffer for re-dissolution, shake and mix, and let it stand at room temperature for 5 minutes. Centrifuge briefly and let it stand on a magnetic rack for 3 minutes. After the liquid is clear, transfer the supernatant to a 1.5 mL centrifuge tube.

[0107] 5.10 After qubit quantification of 1 μL of the purified cDNA product, the fragment distribution was detected using 2100 (as shown in Figure 3). The main cDNA fragment band was approximately 1.2 kb, which is consistent with the cDNA fragment distribution of the existing process.

[0108] 6.cDNA Shearing

[0109] 6.1 Adjust the reaction temperature of a PCR instrument to 55°C and the heating cover temperature to 60°C in advance.

[0110] 6.2 Mix the purified cDNA from 5.9 and transfer 20 ng to a new PCR tube. Add water to 15 μL.

[0111] 6.3 Take 9 μL TE buffer and add 1 μL TME to dilute 10 times, vortex to mix evenly, centrifuge briefly, and set aside.

[0112] 6.4 Prepare the TME disruption mixture in Table 4, vortex to mix evenly, and centrifuge briefly:

[0113] Table 4 TME disruption mixture

[0114] 6.5 Vortex and mix the TME disruption mixture evenly. Centrifuge briefly and place in a 55°C PCR instrument. Cover the PCR instrument and react for 10 minutes.

[0115] 6.6 After the reaction is completed, remove the PCR tube, add 5 μL of stop buffer, vortex to mix, and let it stand at room temperature for 5 minutes.

[0116] 7. PCR and purification

[0117] 7.1 Prepare the PCR Mix according to Table 5;

[0118] Table 5 PCR Mix

[0119] 7.2 Vortex to mix, centrifuge briefly, and place in a PCR instrument. Perform amplification according to the reaction program in Table 6 below.

[0120] Table 6 PCR amplification program

[0121] 7.3 Mix the above PCR product and magnetic beads equilibrated at room temperature at a ratio of 1:0.55 (100 μL PCR product, 55 μL magnetic beads), shake well, and incubate at room temperature for 5 minutes;

[0122] 7.4 After instant centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3-5 minutes. After the liquid becomes clear, carefully pipette the supernatant and transfer it to a new centrifuge tube.

[0123] 7.5 Add 15 μL of magnetic beads to the supernatant, shake to mix, and incubate at room temperature for 5 minutes;

[0124] 7.6 After instant centrifugation, place the tube on a magnetic rack and let it stand for 3-5 minutes until the liquid becomes clear. Use a pipette to carefully remove the supernatant and discard it.

[0125] 7.7 Keep the centrifuge tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol. Rinse the magnetic beads by rotating the centrifuge tube on the magnetic stand. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0126] 7.8 Repeat step 7.7 once;

[0127] 7.9 Try to drain the liquid in the tube. If a small amount of liquid remains on the tube wall, centrifuge the tube instantly, separate it on a magnetic stand, and then use a small-scale pipette to drain the liquid at the bottom of the tube.

[0128] 7.10 Let the magnetic beads air-dry at room temperature for 1-2 minutes until the surface of the beads is free of reflections and cracks.

[0129] 7.11 Add 20 μL of TE buffer for re-dissolution, shake and mix, then let it stand at room temperature for 5 minutes. Centrifuge briefly and let it stand on a magnetic rack for 3 minutes. After the liquid is clear, transfer the supernatant to a 1.5 mL centrifuge tube.

[0130] 7.12 Take 1 μL of the purified product for qubit quantification and then use it for high-throughput sequencing.

[0131] Comparative Example 1 (Constructing spatiotemporal transcriptomes using existing techniques (as shown in FIG1 ))

[0132] In the following process, the chips, RT oligo, and permeabilization reagents were from the Stereo-seq transcriptome reagent set (catalog number: 201ST114), the fragmentation and PCR reagents were from the Stereo-seq library preparation kit (catalog number: 101KL114), the RT buffer and RT enzyme were purchased from BGI (catalog number: LS-EZ-E-00027Q), the RNase inhibitor was purchased from BGI (catalog number: 1000014382), and 25 mM dNTP was purchased from Shanghai Bioengineering (catalog number: B110045-0005).

[0133] The tissue used for constructing the spatiotemporal transcriptome was mouse brain tissue.

[0134] 1. Tissue patching and fixation

[0135] 1.1 Remove the Stereo-seq chip T carrier from the vacuum-dried aluminum foil bag and record the number on the back of the chip;

[0136] Be careful not to touch the front of the chip. Allow to warm to room temperature for 1 minute.

[0137] 1.2 Adjust the PCR instrument temperature to 37°C and the heated cover temperature to 42°C in advance, and place the PCR adapter to balance the temperature.

[0138] 1.3 Pre-cool methanol: Add enough methanol to a slide box or 50 mL centrifuge tube to ensure that the methanol is sufficient to immerse all chips (you can place a regular slide in the container to check whether the volume of methanol is sufficient). Cover the container and pre-cool the methanol at -20°C for 5-30 minutes.

[0139] 1.4 Cut 10 μm tissue slices on a freezing microtome and flatten them. Align the front of the chip with the tissue for mounting.

[0140] 1.5 Quickly place the patched vector on the PCR adapter and incubate at 37°C for 5 minutes to dry.

[0141] 1.6 Immediately place the dried chips in -20°C pre-cooled methanol for 30 minutes to fix them, ensuring that all chips are immersed in methanol.

[0142] 1.7 After fixation, transfer the slide box or 50 mL centrifuge tube to a fume hood;

[0143] 1.8 Remove the carrier from the slide box or 50mL centrifuge tube and use dust-free paper to absorb excess methanol on the back and around the slide to ensure that there is no liquid residue in the gaps between the chips;

[0144] 1.9 Place the carrier upright on a slide staining rack and ventilate in a fume hood for 4-6 minutes to allow the methanol to evaporate completely;

[0145] 1.10 After the methanol evaporates and the tissue turns white, transfer the carrier to the lab table. Assemble the gasket and fixture to form a carrier, and secure the chip to the carrier to form a handheld carrier.

[0146] 2. Tissue Permeabilization

[0147] 2.1 Prepare 1× Permeabilization Reagent Working Solution by preparing 2 mL of 0.01 N HCl in advance. Dissolve PR Enzyme (red cap, powdered) in 1 mL of freshly prepared 0.01 N HCl and mix thoroughly by pipetting. Then, dilute 10 μL of the 10× Permeabilization Reagent Stock Solution to 100 μL with 0.01 N HCl. Label this solution as 1× Permeabilization Reagent Working Solution.

[0148] 2.2 Set the temperature of two PCR machines (or one PCR machine and one metal bath) to 37°C and the heated cover temperature to 42°C in advance. Place a PCR adapter in one of the PCR machines to balance the temperature.

[0149] 2.3 Place the handheld carrier on the PCR adapter, cover the PCR instrument, and rewarm at 37°C for 3 minutes. At the same time, place the 1× permeabilization reagent working solution in the PCR instrument or metal bath and rewarm at 37°C for 3 minutes.

[0150] 2.4 After rewarming, add 1× permeabilization reagent working solution to one corner of the chip at a volume of 100 μL / chip. Seal the handheld carrier with sealing film and cover the PCR instrument.

[0151] 2.5 Incubate the permeabilization reaction at 37°C for 11 minutes.

[0152] 3. Reverse transcription (the reverse transcription process in existing spatiotemporal transcriptomics technology, the reagents of which are all from the Stereo-seq transcriptomics reagent kit (Cat. No.: 201ST114)).

[0153] 3.1 Prepare RT reagent, RT additive and RT oligo according to the BGI Stereo-seq transcriptome reagent kit (Cat. No. 201ST114), dissolve and place on ice;

[0154] 3.2 While waiting for permeabilization, prepare the RT reaction mixture according to Table 7 and place it on ice;

[0155] Table 7 RT reaction mixture (RT Mix) in the prior art

[0156] 3.3 During permeabilization, adjust the reaction temperature of another PCR instrument to 42°C and the heated lid temperature to 47°C. Place another PCR adapter to balance the temperature.

[0157] 3.4 Remove the permeabilized handheld carrier from the PCR instrument;

[0158] 3.5 Slightly tilt the handheld carrier to an angle of less than 20° and use a pipette to remove the permeabilization reagent from one corner of the chip;

[0159] Note: When tearing off the sealing film, do not press the upper part of the clamp buckle on both sides to prevent the carrier from falling off.

[0160] 3.6 Add 100 μL of PR Rinse Buffer solution;

[0161] 3.7 Slightly tilt the handheld carrier to an angle of less than 20° and use a pipette to aspirate the PR Rinse Buffer solution at one corner of the chip to keep the chip moist.

[0162] NOTE: Avoid allowing the chip to dry out completely.

[0163] 3.8 Take out the prepared RT Mix, pipette and mix thoroughly, then centrifuge briefly. Add RT Mix to one corner of the chip to ensure that the RT Mix evenly covers the entire chip. The reverse transcription primer loaded on the chip is the same as in Example 1.

[0164] 3.9 Seal the handheld carrier with a sealing film, place it on the PCR adapter of a 42°C PCR instrument, close the PCR instrument lid, and react for 3 hours.

[0165] 4. Tissue Removal

[0166] 4.1 Prepare reagents;

[0167] Table 8:

[0168] Note: If a white precipitate is observed in the buffer, it can be dissolved at 55°C and then returned to room temperature.

[0169] 4.2 Adjust the reaction temperature of another PCR instrument to 55°C in advance, the temperature of the PCR instrument's heated cover to 60°C, and place the PCR adapter to balance the temperature;

[0170] Table 9:

[0171] 4.3 Remove the handheld carrier from the PCR instrument (42°C);

[0172] 4.4 Slightly tilt the handheld carrier and use a pipette to aspirate the RT Mix on the chip surface;

[0173] 4.5 Add TR Buffer (400 μL / well) and place on the PCR adapter of a PCR instrument (55°C) for 10 minutes. Note: cDNA Release Mix can be prepared in advance for this step.

[0174] 4.6 Slightly tilt the handheld carrier and aspirate the TR Buffer. Note: If the tissue is not removed cleanly, add 400 μL of 0.1× SSC and gently pipette to remove the tissue from the chip. Then, slightly tilt the handheld carrier and aspirate the 0.1× SSC.

[0175] 5.cDNA Release and Recovery

[0176] 5.1 Prepare cDNA Release Mix according to Table 10 and store at room temperature;

[0177] 5.2 Add cDNA Release Mix (400 μL / well);

[0178] 5.3 Seal the handheld carrier with a plate sealer to prevent evaporation. Place the carrier on the PCR adapter of a PCR instrument (55°C) and allow the reaction to proceed for 3 hours or more (as shown in Table 10), but no longer than 18 hours. Elute the first strand of the cDNA.

[0179] Table 10 cDNA Release Mix

[0180] Table 11:

[0181] 5.4 After the reaction is completed, completely recover the liquid in the reaction well into a new 1.5 mL centrifuge tube;

[0182] 5.5 Add Nuclease Free Water to clean the reaction well chip (100 μL / well) and collect the liquid into the same 1.5 mL centrifuge tube.

[0183] 6.cDNA Purification and Amplification

[0184] 6.1 cDNA purification;

[0185] If a white precipitate is observed in the cDNA recovery solution, dissolve it at 55°C and return it to room temperature before purification. Remove the magnetic beads 30 minutes in advance and equilibrate them at 37°C (this equilibration temperature only applies to VAHTS™ DNA Clean Beads).

[0186] b.1.0× magnetic bead cDNA purification steps:

[0187] 1) Mix the recovery solution (450-490 μL) from the previous step with the magnetic beads equilibrated at room temperature in a ratio of 1:1, shake to mix, and incubate at room temperature for 10 minutes;

[0188] 2) After instant centrifugation, place the centrifuge tube on a magnetic stand and let it stand for 3 minutes;

[0189] 3) Once the liquid is clear, carefully remove the supernatant with a pipette (if there is foam on the tube cap, discard it).

[0190] 4) Place the centrifuge tube on the magnetic rack and add 1 mL of 80% ethanol (use freshly prepared 80% ethanol equilibrated to room temperature). Rotate the tube on the magnetic rack until all the magnetic beads are adsorbed to the side of the tube closest to the magnetic rack. Rotate the tube again to allow the beads to re-adsorb to the other side to rinse the beads. Let it sit for 30 seconds, then carefully aspirate and discard the supernatant. Keep the pipette tip against the tube away from the magnetic rack. Do not agitate or disturb the beads. (If there is foam on the tube cap, it is recommended to clean it with 80% ethanol.)

[0191] 5) Repeat step 4 once;

[0192] 6) Place the centrifuge tube on the magnetic rack and air-dry at room temperature for 5-8 minutes until the surface of the magnetic beads is free of reflections and cracks.

[0193] 7) First, add 22 μL of Nuclease-Free Water to dissolve the solution, shake and mix, then let it stand at room temperature for 5 minutes, centrifuge briefly, and place it on a magnetic stand for 3-5 minutes until the liquid becomes clear;

[0194] 8) Transfer the supernatant (~21 μL cDNA) to a new 0.2 mL PCR tube;

[0195] 9) Add 22 μL of Nuclease-Free Water to the magnetic beads from step 7 for a second re-dissolution. Mix thoroughly by vortexing and let stand at room temperature for 5 minutes. Centrifuge briefly and place on a magnetic stand for 3-5 minutes until the liquid becomes clear.

[0196] 10) Transfer the supernatant (~21 μL cDNA) to the PCR tube indicated in step 9, for a total volume of 42 μL. If the recovered sample volume is less than 42 μL, add Nuclease Free Water to make up the volume.

[0197] 6.2 cDNA amplification;

[0198] a. Prepare 100 μL of PCR Mix according to Table 12 (all reagents are from the Stereo-seq Transcriptome Reagent Set (BGI, Cat. No. 201ST114)).

[0199] Table 12 PCR Mix

[0200] b. Centrifuge briefly and amplify according to the PCR program in Table 13:

[0201] Table 13 PCR amplification program (reaction system 100 μL)

[0202] c. Take 1 μL of PCR product for qubit quantification and record the concentration;

[0203] d. Purify the PCR product with 1.0× magnetic beads;

[0204] 1) Mix PCR product (100 μL) with magnetic beads equilibrated at room temperature at a ratio of 1:1, shake to mix, and incubate at room temperature for 10 minutes;

[0205] 2) After centrifugation, place the PCR tube on a magnetic rack for 3 minutes. Once the liquid is clear, remove the supernatant.

[0206] 3) Keep the tube on the magnetic rack and rinse with 200 μL of 80% ethanol (freshly prepared and equilibrated to room temperature). Rinse the beads by rotating the tube on the magnetic rack. Let it sit for 30 seconds, then carefully aspirate and discard the supernatant. Keep the pipette tip against the tube wall away from the magnetic rack. Do not agitate or disturb the beads.

[0207] 4) Repeat step 3 once;

[0208] 5) Place the centrifuge tube on the magnetic rack, open the lid, and air-dry at room temperature for 5-8 minutes until the surface of the magnetic beads is free of reflections and cracks.

[0209] 6) Add 42 μL of TE to dissolve the sample, vortex to mix, and let it stand at room temperature for 5 minutes. Centrifuge briefly and place on a magnetic rack for 3-5 minutes. Once the liquid has clarified, transfer the supernatant (~40 μL) to a new 1.5 mL centrifuge tube. (You can stop at this step and store the sample at -20°C.)

[0210] 7) Take 1 μL of the purified cDNA product for qubit quantification and use 2100 to detect the fragment distribution, as shown in Figure 4.

[0211] 7.cDNA Shearing

[0212] 7.1 Adjust the reaction temperature of a PCR instrument to 55°C and the heating cover temperature to 60°C in advance.

[0213] 7.2 Mix the purified cDNA from 5.9 and transfer 20 ng to a new PCR tube. Add water to 15 μL.

[0214] 7.3 Take 9 μL TE buffer and add 1 μL TME to dilute 10 times, vortex to mix evenly, centrifuge briefly, and set aside.

[0215] 7.4 Prepare the TME disruption mixture in Table 14, vortex to mix evenly, and centrifuge briefly:

[0216] Table 14 TME disruption mixture

[0217] 7.5 Vortex and mix the TME disruption mixture evenly. Centrifuge briefly and place in a 55°C PCR instrument. Cover the PCR instrument lid and react for 10 minutes.

[0218] 7.6 After the reaction is completed, remove the PCR tube, add 5 μL of stop buffer, vortex to mix, and let it stand at room temperature for 5 minutes.

[0219] 8.PCR and purification

[0220] 8.1 Prepare the PCR Mix according to Table 15;

[0221] Table 15 PCR Mix

[0222] 8.2 Vortex to mix, centrifuge briefly, and place in a PCR instrument. Perform amplification according to the reaction program in Table 16.

[0223] Table 16 PCR amplification program

[0224] 8.3 Mix the above PCR product and magnetic beads equilibrated at room temperature at a ratio of 1:0.55 (100 μL PCR product, 55 μL magnetic beads), shake well, and incubate at room temperature for 5 minutes;

[0225] 8.4 After instant centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3-5 minutes. After the liquid becomes clear, carefully pipette the supernatant and transfer it to a new centrifuge tube.

[0226] 8.5 Add 15 μL of magnetic beads to the supernatant, shake to mix, and incubate at room temperature for 5 minutes;

[0227] 8.6 After a quick centrifugation, place the tube on a magnetic rack and let it sit for 3-5 minutes until the liquid becomes clear. Carefully remove the supernatant with a pipette and discard.

[0228] 8.7 Keep the centrifuge tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol. Rinse the magnetic beads by rotating the centrifuge tube on the magnetic stand. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0229] 8.8 Repeat step 8.7 once;

[0230] 8.9 Try to drain the liquid in the tube. If a small amount of liquid remains on the tube wall, centrifuge the tube instantly, separate it on a magnetic stand, and then use a small-scale pipette to drain the liquid at the bottom of the tube.

[0231] 8.10 Let the magnetic beads air-dry at room temperature for 1-2 minutes until the surface of the beads is free of reflections and cracks.

[0232] 8.11 Add 20 μL of TE buffer for re-dissolution, shake and mix, then let it stand at room temperature for 5 minutes. Centrifuge briefly and let it stand on a magnetic rack for 3 minutes. After the liquid is clear, transfer the supernatant to a 1.5 mL centrifuge tube.

[0233] 8.12 Take 1 μL of the purified product for qubit quantification and then use it for high-throughput sequencing.

[0234] Except for the different values ​​of dNTPs, template switching primers, and reverse transcriptase in "3. Reverse Transcription", the other steps of Example 2-6 and Comparative Example 2-5 are the same as those of Example 1 (see Table 18 for details).

[0235] Experimental testing:

[0236] The number of gene detections in individual cells of the spatiotemporal transcriptomes constructed in Examples 1-7 and Comparative Examples 1-5 was tested, and the specific results are shown in Tables 17 and 18. Bin200 Median MID(K), Bin20 Median Gene, and Bin200 Median Gene(K) represent the MID and gene median under different bins, respectively. A higher number of genes indicates more genes captured during the experiment, that is, better library quality. There is a positive correlation between these three.

[0237] Table 17 Comparison of the spatiotemporal transcriptome process in Example 1 and Comparative Example 1

[0238] Table 18 Comparison of the spatiotemporal transcriptome process in Examples 2-7 and Comparative Examples 2-5

[0239] From the above description, it can be seen that the above-mentioned embodiment of the present invention achieves the following technical effects: in the process of cDNA synthesis, the reverse transcription mixture of the present application is used to perform the reverse transcription reaction, and the efficiency of the reverse transcription reaction is improved by optimizing the amount of dNTPs, template conversion primers and reverse transcriptase involved in the reaction in the reverse transcription reaction system. It is possible to obtain a double-stranded cDNA product in a one-step reaction, greatly shortening the reaction time of reverse transcription and improving the quality of the product cDNA. It also improves the quality of library construction in technologies such as spatiotemporal transcriptome, conventional RNA transcriptome, and single-cell transcriptome that require double-stranded cDNA synthesis, increases the number of gene detections in single cells of the spatiotemporal transcriptome, and thus obtains more accurate sequencing results.

[0240] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for synthesizing double-stranded cDNA, characterized in that: The method comprises: Performing a reverse transcription reaction on the mRNA template using a reverse transcription primer and a reverse transcription mixture to obtain the cDNA double strand; Wherein, the reverse transcription mixture comprises a reverse transcriptase with template switching function, a template switching primer and dNTPs; In the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

2. The method according to claim 1, characterized in that The final concentration of the reverse transcriptase is 8-12 U / μL, the final concentration of the template switching primer is 2.5-5 μM, and the final concentration of the dNTPs is 4-6 mM; Preferably, the final concentration of the reverse transcription primer is 0.5-5 μM; Preferably, the reverse transcription mixture further comprises: a reverse transcription buffer and an RNase inhibitor.

3. The method according to claim 1 or 2, characterized in that The reverse transcription reaction comprises: synthesizing a first-strand cDNA from the mRNA template using the reverse transcription primer and the reverse transcriptase to obtain a first-strand cDNA product bound to the template switching primer; Reversely synthesizing the first-strand cDNA product using the template switching primer and the reverse transcriptase to obtain the double-stranded cDNA; Preferably, the reaction temperatures for the first-strand cDNA synthesis and the second-strand cDNA synthesis are each independently 42-50°C.

4. The method according to claim 1, wherein The template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, the N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; Preferably, the length of the template switching primer is 20-35 nt; Preferably, the mRNA template is derived from tissue or cells.

5. A method for constructing a spatiotemporal transcriptome library, characterized in that: The method comprises: The mRNA template is combined with the reverse transcription primer fixed on the spatiotemporal chip, and a reverse transcription mixture is added to perform a reverse transcription reaction to obtain a double-stranded cDNA; Dissociating the cDNA double strands to obtain a free cDNA second strand; Performing a PCR reaction using the free cDNA second strand as a template to obtain a free cDNA double strand; constructing a fragmentation library of the free cDNA double strands to obtain the spatiotemporal transcriptome library; The reverse transcription mixture includes a reverse transcriptase with template switching function, a template switching primer and dNTPs; In the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switching primer is 2-12.5 μM, and the final concentration of the dNTPs is 1-10 mM.

6. The method according to claim 5, characterized in that The final concentration of the reverse transcriptase is 8-12 U / μL, the final concentration of the template switching primer is 2.5-5 μM, and the final concentration of the dNTPs is 4-6 mM; Preferably, the final concentration of the reverse transcription primer is 0.5-5 μM; Preferably, the reverse transcription mixture further comprises: a reverse transcription buffer and an RNase inhibitor.

7. The method according to claim 5 or 6, characterized in that The reverse transcription reaction comprises: The reverse transcription primer binds to the mRNA template to perform a reverse transcription reaction of the first strand of cDNA, thereby obtaining a first strand of cDNA product that binds to the template switching primer; The template switching primer and the reverse transcriptase reversely synthesize the cDNA second strand of the cDNA first strand product to obtain the cDNA double strand; Preferably, the dissociation comprises alkaline lysis of the cDNA double strands and neutralization of the resulting lysate, wherein the lysate comprises a free cDNA second strand; Preferably, the reaction temperatures for the first-strand cDNA synthesis and the second-strand cDNA synthesis are each independently 42-50°C.

8. The method according to claim 5, characterized in that The template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, the N is any one of the bases A, T, U, G or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; Preferably, the length of the template switching primer is 20-35 nt; Preferably, the mRNA template is derived from tissue or cells.

9. A kit, characterized in that The kit includes a reverse transcriptase with a template switching function, a template switching primer, a reverse transcription primer and dNTPs; Wherein, the working concentration of the reverse transcriptase is 5-20 U / μL, the working concentration of the template switching primer is 2-12.5 μM, and the working concentration of the dNTPs is 1-10 mM; Preferably, the working concentration of the reverse transcriptase is 8-12 U / μL, the working concentration of the template switching primer is 2.5-5 μM, and the working concentration of the dNTPs is 4-6 mM; Preferably, the working concentration of the reverse transcription primer is 0.5-5 μM; Preferably, the kit further comprises: a reverse transcription buffer and an RNase inhibitor; Preferably, the template switching primer includes: a template switching primer whose first three nucleotides at the 3' end are NGG or NNN, wherein the nucleotides include ribonucleotides and deoxyribonucleotides, the N is any one of the bases A, T, G, U or C, and the third nucleotide from the 3' end has a locked nucleic acid modification; Preferably, the length of the template switching primer is 20-35 nt; Preferably, the mRNA template is derived from tissue or cells.

10. A RT-qPCR detection method, characterized in that: The detection method comprises: Reverse transcription of the test RNA using the method according to any one of claims 1 to 4 to obtain a double-stranded cDNA; The fluorescent PCR reaction is performed using the cDNA double strand as a template.

11. A method for constructing a transcriptome library, characterized in that: The method comprises: Reverse transcription of the test RNA using the method according to any one of claims 1 to 4 to obtain a double-stranded cDNA; The cDNA double strands are fragmented to construct a library to obtain the transcriptome library.

12. The method according to claim 11, characterized in that The transcriptome library includes a single-cell transcriptome library.

13. A sequencing method, characterized in that: The method comprises: The spatiotemporal transcriptome library constructed by the method for constructing a spatiotemporal transcriptome library according to any one of claims 5 to 8, or the transcriptome library obtained by the method according to claim 11 or 12, is sequenced to obtain a sequencing result.

14. Use of the kit according to claim 9 in double-stranded cDNA synthesis.

Images