mRNA enrichment method

Abstract

Provided is a mRNA enrichment method, relating to the technical field of biology. According to the method, first capture magnetic beads and second capture magnetic beads are respectively used for carrying out two rounds of capturing on mRNAs, such that rRNA residues in a product can be effectively reduced, and a high-purity mRNA product is obtained, thereby improving the data validity of downstream transcription analysis.

Description

A method for mRNA enrichment Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for enriching mRNA. Background Art

[0002] In the past decade, transcriptome sequencing (RNA-seq) has become an important tool for analyzing differential gene expression and differential splicing of mRNAs across the entire transcriptome. The transcriptome refers to the collection of all transcripts in a cell, tissue, or organism under a specific physiological condition, that is, the sum of all transcribed RNAs, including different types of RNA molecules such as coding RNA (mRNA) and non-coding RNA (tRNA, rRNA, miRNA, lncRNA, circRNA). Among them, rRNA accounts for more than 80% of the total RNA and is the most abundant type of RNA (Stark R, Grzelak M, Hadfield J. RNA sequencing: the teenage years. Nat Rev Genet. 2019 Nov; 20(11): 631-656.). As the most abundant member of total RNA, rRNA provides very little transcript information, and excessive detection will mask the expression abundance of other genes, thereby increasing the difficulty of detecting low-abundance transcripts. Therefore, rRNA is usually removed from total RNA samples before sequencing, and the efficiency of rRNA removal is also considered a key factor in maximizing the readout of transcripts.

[0003] Currently, most published RNA-seq data are based on oligo(dT)-enriched mRNA. This enrichment method selects transcripts containing poly(A) tails and focuses on sequencing the protein-coding regions of the transcriptome. However, currently known mRNA extraction schemes have very inconsistent rRNA residue performance across different species. For example, while they can achieve good enrichment results in animal tissues, with rRNA residues below 5%, they perform poorly in different plants and fungi, with some rRNA residues reaching as high as 90%. This significantly limits the effectiveness of transcriptome analysis.

[0004] Summary of the Invention

[0005] The purpose of this application is to provide an mRNA enrichment method that can significantly reduce rRNA residue. This method is based on existing technologies and is optimized. During the second mRNA enrichment, a second capture magnetic bead is used to capture the mRNA, allowing the mRNA to bind to the new capture magnetic beads. This can effectively reduce rRNA residue in the product and increase the proportion of mRNA, thereby increasing the detection efficiency of low-abundance transcripts and the effectiveness of transcriptional analysis.

[0006] The first aspect of the present application provides a method for mRNA enrichment, comprising the following steps:

[0007] (1) mixing the total RNA sample with first capture magnetic beads containing Oligo(dT) and incubating;

[0008] (2) Apply an external magnetic field and remove the supernatant;

[0009] (3) washing the first capture magnetic beads and removing the washing solution;

[0010] (4) eluting the mRNA from the first capture magnetic beads to obtain an elution product;

[0011] (5) applying an external magnetic field to transfer the eluted product and discarding the first capture magnetic beads, adding second capture magnetic beads to the eluted product and incubating, wherein the second capture magnetic beads contain Oligo(dT);

[0012] (6) Apply an external magnetic field and remove the supernatant;

[0013] (7) washing the second capture magnetic beads and removing the washing solution;

[0014] (8) Elute the mRNA from the second capture magnetic beads.

[0015] In some embodiments, the surfaces of the first and second capture magnetic beads are coupled with oligothymidine nucleotide chains (Oligo(dT)), which complementarily bind to the PolyA tail of mRNA, and the number of nucleotides in the oligothymidine nucleotide chains is 5-50, preferably 5-30, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 within the range.

[0016] In some embodiments, the surface of each of the first capture magnetic beads and each of the second capture magnetic beads is coupled with at least one oligothymidine nucleotide chain (Oligo(dT)), for example, at least 10, at least 100, at least 1000, at least 5000, at least 10000, at least 20000, at least 50000 and at least 100000.

[0017] In some embodiments, the incubation temperature of step (1) is 50-80°C, preferably 55-75°C, including 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C and 75°C within the range.

[0018] In some embodiments, the incubation time of step (1) is 1-10 minutes, including 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes and 10 minutes within the range.

[0019] In some embodiments, the incubation conditions of step (1) are, for example, incubation at 50-80°C for 1-10 minutes, for example, incubation at 50-75°C for 1-10 minutes, for example, incubation at 55-75°C for 1-10 minutes, for example, incubation at 55-75°C for 1-8 minutes, for example, incubation at 55-75°C for 2-8 minutes, for example, incubation at 60-75°C for 2-8 minutes, for example, incubation at 60-75°C for 3-8 minutes, for example, incubation at 60-70°C for 3-8 minutes, for example, incubation at 60-70°C for 3-7 minutes, for example, incubation at 60-70°C for 3-6 minutes, for example, incubation at 62-68°C for 3-6 minutes, for example, incubation at 62-68°C for 4-6 minutes, for example, incubation at 65°C for 5 minutes, including but not limited to any combination of the foregoing incubation temperatures and incubation times.

[0020] In some embodiments, the incubation conditions of step (1) include incubating at 50-80°C for 1-10 minutes and then incubating at room temperature for 1-10 minutes; preferably, the room temperature incubation is 1-10 minutes, such as 2-8 minutes, such as 2-7 minutes, such as 3-7 minutes, such as 4-6 minutes, such as 5 minutes. The room temperature is room temperature 20-35°C, including 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C and 35°C within the range.

[0021] In some embodiments, the incubation conditions of step (1) include incubation at 65° C. for 5 minutes and incubation at room temperature for 5 minutes.

[0022] In some embodiments, the incubation conditions of step (5) include incubation at room temperature for 1-10 minutes, for example, 2-8 minutes, for example, 2-7 minutes, for example, 3-7 minutes, for example, 4-6 minutes, for example, 5 minutes, and the room temperature is room temperature 20-35°C, including 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C and 35°C within the range.

[0023] In some embodiments, the incubation conditions of step (5) include incubating at 50-80°C for 1-10 minutes and then incubating at room temperature for 1-10 minutes; for example, incubating at 60-70°C for 1-10 minutes and then incubating at room temperature for 1-10 minutes; for example, incubating at 60-70°C for 3-8 minutes and then incubating at room temperature for 3-8 minutes; for example, incubating at 65°C for 5 minutes and then incubating at room temperature for 5 minutes.

[0024] In some embodiments, the external magnetic field in step (2), step (5) and step (6) is, for example, placing the sample on a magnetic stand.

[0025] In some embodiments, the washing comprises adding a washing solution, for example, adding a washing solution to resuspend the magnetic beads and mixing by pipetting. In some embodiments, the washing solution includes but is not limited to all magnetic bead washing solutions known in the art, for example, the washing solution comprises salt and buffer components. In some embodiments, the washing temperature is room temperature, for example, room temperature 20-35°C, including 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C and 35°C within the range.

[0026] In some embodiments, the washing may be repeated once or twice or more.

[0027] In some embodiments, the elution comprises adding an elution solution, which includes but is not limited to any magnetic bead elution solution known in the art, such as pure water or Tris buffer, and the elution temperature is 50-90° C., such as 50-85° C., such as 60-85° C., such as 70-85° C., such as 75-80° C., such as 80° C. In some embodiments, the elution comprises incubating for 1-10 minutes, such as 1-8 minutes, such as 1-6 minutes, such as 1-6 minutes, such as 1-3 minutes, such as 2 minutes.

[0028] In some embodiments, the total RNA sample is from a eukaryotic organism, including but not limited to animals, plants, protists, and fungi.

[0029] In some embodiments, the animals include but are not limited to humans, mice, rats, guinea pigs, rabbits, Dogs, monkeys, cows, pigs, fish, turtles, cats, sheep, pigeons, chickens, frogs, toads, horses, crab-eating macaques, rhesus macaques, orangutans, snakes, baboons, marmosets, cotton rats and ferrets; the fungi include but are not limited to yeast, rust, white mold, streptomyces, aflatoxin, Penicillium, graminearum, dahliae, Brucella, Aspergillus oryzae, graminearum, Aspergillus, anthrax, Fusarium, flax rust, Cryptococcus, Aspergillus fumigatus, auris, Mucor, Candida albicans, ergot, Chaetomium and mushrooms, etc., the mushrooms are for example shiitake mushrooms, straw mushrooms, wood ear, white ear, hericium erinaceus, bamboo fungus, Agaricus bisporus, matsutake, ganoderma, cordyceps, truffles, boletus, morels and saddle mushrooms, etc.; the protozoa include but are not limited to paramecium, amoeba, plasmodium, The plants are, for example, seed plants or spore plants, including but not limited to rice, Arabidopsis, tomato, wheat, camphor tree, carpet grass, raspberry, strawberry, apple, fir, pittosporum, linden, cnidium, locust tree, iron tree, tea tree, dandelion, soapberry, azalea, Podocarpus, palm, ginkgo, garlic, pear, cherry, potato, areca palm, peach, forsythia, osmanthus tree, longan, mango, oak, loquat, bamboo, wolfberry, apricot, crabapple, persimmon, coconut palm, monstera, spring feather, lychee, sumac, sugarcane, locust tree, rose, elm, wisteria tree, birch, chrysanthemum, rapeseed, soybean, algae, fungi, lichen, moss and pteridophyte, and the sampling parts of the plants are, for example, leaves, seeds, roots, flowers, fruits and stems.

[0030] The second aspect of the present application provides a method for constructing a transcriptome library, the method comprising the following steps:

[0031] (1) capturing mRNA using the method described in the first aspect;

[0032] (2) reverse transcribing the mRNA to synthesize a first-strand cDNA; optionally, synthesizing a second-strand cDNA after the synthesis of the first-strand cDNA;

[0033] (3) adding the transposase complex and incubating to obtain fragmented cDNA;

[0034] (4) adding primers with linker sequences and obtaining amplified products with linker sequences by PCR amplification;

[0035] (5) Optionally, purify the amplified product.

[0036] In some embodiments, the reverse transcription uses M-MLV reverse transcriptase or AMV reverse transcriptase, preferably M-MLV reverse transcriptase.

[0037] In some embodiments, the primer used in the reverse transcription is an Oligo(dT) primer or a random hexamer, preferably a random hexamer.

[0038] In some embodiments, the transposase complex comprises a transposase and a transposon.

[0039] In some embodiments, the transposon comprises a transposase recognition core sequence, such as an OE sequence or a ME sequence.

[0040] In some embodiments, the transposon further includes a sequencing primer sequence, which is single-stranded or double-stranded. The sequencing primer sequence can be one or two, such as a read1 sequence and a read2 sequence of an Illumina platform.

[0041] In some embodiments, the transposase binds to the transposon to form a transposase complex.

[0042] In some embodiments, the two transposase complexes synapse, and the ends interact and dimerize to form a synaptic transposase complex consisting of a dimeric protein and two transposon molecules, wherein the two transposon molecules may or may not contain a sequencing primer sequence. In some embodiments, the two transposon molecules contain the same or different sequencing primer sequences.

[0043] In some embodiments, the amplification primer comprises a sequence that is identical or complementary to a transposase recognition core sequence.

[0044] In some embodiments, the amplification primer further comprises a sequence complementary to a sequencing primer sequence.

[0045] In some embodiments, the amplification primer further comprises an index sequence. In some embodiments, the index sequence is, for example, 6-8 bases, preferably 8 bases.

[0046] In some embodiments, the amplification primers are amplification primers with adapter sequences, and the amplification primers include a first primer with a first adapter sequence and a second primer with a second adapter sequence.

[0047] In some embodiments, the adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the ion torrent platform, the Illumina platform, and the BGI platform).

[0048] In some embodiments, the first adapter sequence is a P5 adapter sequence of an Illumina platform, and the second adapter sequence is a P7 adapter sequence of an Illumina platform, or vice versa.

[0049] In some embodiments, the first adapter sequence is a P1 adapter sequence of an ion torrent platform and the second adapter sequence is an A adapter of an ion torrent platform, or vice versa.

[0050] In some embodiments, the first adapter sequence is an upstream splint sequence for single-ended tag library construction on the MGI platform, and the second adapter sequence is a downstream splint sequence for single-ended tag library construction on the MGI platform, or vice versa. In some embodiments, the first adapter sequence is an upstream splint sequence for double-ended tag library construction on the MGI platform, and the second adapter sequence is a downstream splint sequence for double-ended tag library construction on the MGI platform, or vice versa.

[0051] In some embodiments, the first primer is an N5 primer and the second primer is an N7 primer, or vice versa.

[0052] In some embodiments, the transposase is a Tn5 transposase, which is any Tn5 transposase known in the art or an active mutant thereof.

[0053] In some embodiments, the Tn5 transposase is a Tn5 transposase having the activity of breaking RNA-cDNA hybrid chains.

[0054] In some embodiments, the method further comprises a step of terminating the fragmentation reaction, eg, by inactivating the transposase, eg, by adding EDTA.

[0055] In some embodiments, the purification step uses a magnetic bead method, a high salt precipitation method, a centrifugal column method, or a phenol-chloroform extraction method, preferably a magnetic bead method.

[0056] The third aspect of the present application provides a method for constructing a transcriptome library, the method comprising the following steps:

[0057] (1) capturing mRNA using the method described in the first aspect;

[0058] (2) interruption to obtain fragmented mRNA;

[0059] (3) reverse transcribing the fragmented mRNA to synthesize cDNA;

[0060] (4) performing end repair on the cDNA;

[0061] (5) Connector connection;

[0062] (6) Library amplification.

[0063] In some embodiments, the fragmentation includes but is not limited to fragmenting the mRNA using a fragmentation enzyme method, a chemical method, and / or a mechanical method. In some embodiments, the mechanical method is, for example, ultrasonic fragmentation, centrifugation, or heat shock, and the chemical method is, for example, treating the mRNA with a chemical reagent to break it into multiple fragments. In some embodiments, the fragmentation enzyme method is, for example, using one or more of endonuclease V, deoxyribonuclease I, and T7 endonuclease I.

[0064] In some embodiments, the reverse transcription uses M-MLV reverse transcriptase or AMV reverse transcriptase, preferably M-MLV reverse transcriptase. In some embodiments, the primer used in the reverse transcription is an Oligo (dT) primer or a random hexamer, preferably a random hexamer.

[0065] In some embodiments, the step (2) and the step (3) can be combined into one step, or the step (3) and the step (4) can be combined into one step, or the step (4) and the step (5) can be combined into the same step.

[0066] In some embodiments, the cDNA is double-stranded cDNA.

[0067] In some embodiments, the end repair comprises blunting the ends of the cDNA to enable ligation of the cDNA to blunt-ended adapters.

[0068] In some embodiments, the end repair further comprises the step of adding A, ie, adding adenine deoxyribonucleotide to the 3' end of the cDNA after the end is blunted, so as to connect the cDNA to the linker via AT.

[0069] In some embodiments, the enzyme used to blunt-end the cDNA is, for example, T4 DNA polymerase, and the enzyme used in the A addition step is, for example, Klenow DNA polymerase (3'-5'exo-).

[0070] In some embodiments, the adapter is a Y-type adapter of the Illumina platform, a bubble adapter of the MGI platform, or a sequencing adapter of the Ion Torrent platform.

[0071] In some embodiments, the linker ligation uses T4 DNA ligase.

[0072] In some embodiments, the method further comprises purifying the product of library amplification, wherein the purification adopts a magnetic bead method, a high salt precipitation method, a centrifugal column method or a phenol-chloroform extraction method, preferably a magnetic bead method.

[0073] A fourth aspect of the present application provides a transcriptome analysis method, comprising the following steps:

[0074] (1) constructing a library using the method described in the second aspect or the third aspect;

[0075] (2) Sequencing.

[0076] A fifth aspect of the present application provides a method for detecting mRNA expression, the method comprising:

[0077] (1) capturing mRNA using the method described in the first aspect;

[0078] (2) Perform qRT-PCR detection.

[0079] The sixth aspect of the present application provides the use of capture magnetic beads in reducing rRNA residues, wherein the capture magnetic beads comprise first capture magnetic beads and second capture magnetic beads, and the first capture magnetic beads and the second capture magnetic beads are coupled to oligothymidine nucleotide chains (Oligo(dT)).

[0080] The seventh aspect of the present application provides use of the method of the first aspect in reducing rRNA residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1: Comparison of the operational procedures of the prior art (one-round grasping) and the technical solution of the present application (two-round grasping);

[0082] Figure 2: RRNA ratios detected by the two protocols when the sample input was 200 ng;

[0083] Figure 3: RRNA ratios detected by the two protocols when the sample input was 500 ng;

[0084] Figure 4: mRNA ratios detected by the two protocols when the sample input was 200 ng;

[0085] Figure 5: mRNA ratios detected by the two protocols when the sample input was 500 ng;

[0086] Figure 6: Number of genes detected by the two protocols when the sample input was 200 ng;

[0087] Figure 7: Number of genes detected by the two protocols when the sample input was 500 ng;

[0088] Figure 8: RRNA ratios detected by the two protocols using Yeasen mRNA capture beads with a sample input of 200 ng.

[0089] Figure 9: RRNA ratios detected by the two protocols using Yeasen mRNA capture beads with a sample input of 500 ng.

[0090] Figure 10: 11 different samples, rRNA ratios detected using two protocols at an input of 500 ng;

[0091] Figure 11: Operational flow of the optimized two-wheeled grasping technology solution;

[0092] Figure 12: The proportion of rRNA detected using two two-round extraction schemes when the sample input is 500 ng.

[0093] Specific implementation (embodiment)

[0094] The technical solution of the present application is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0095] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0096] Example 1

[0097] 1. Total RNA extracted from 293 cells, tomato fruits, Verticillium dahliae, and wheat seeds was used as samples, and the protocol was tested according to the instructions for Vazyme mRNA capture magnetic beads with the product number N403. The magnetic beads in N403 have Oligo (dT), which can capture mRNA through AT complementarity. The total RNA input was 200ng and 500ng, respectively, and the two experimental schemes shown in Figure 1 were used respectively. One round of capture was a conventional method in the current prior art (the process on the left side of Figure 1). After the mRNA was eluted for the first time, a magnetic bead binding solution was directly added to the eluted product, allowing the magnetic beads to capture the mRNA for a second time, that is, the magnetic beads used for the second capture were the magnetic beads released in the previous step); the process on the right side of Figure 1 is the two-round capture method of the present application, which adds the steps of transferring the supernatant and replacing new magnetic beads when capturing mRNA for the second time.

[0098] 2. RNA-seq library was constructed for the above products. The specific process was carried out according to the instructions of Vazyme#NR605.

[0099] 3. The constructed RNA-seq library was sequenced using PE150. The rRNA residues in the sequencing reads were counted, as shown in Figures 2-3. The mRNA ratios in the sequencing reads were counted, as shown in Figures 4-5. The number of gene calls in the sequencing results was counted, as shown in Figures 6-7. The "Expression Gene" on the vertical axis refers to the number of genes mapped to the mRNA reads.

[0100] The results showed that the innovative two-round extraction method can significantly reduce rRNA residue. As shown in Figure 3, when the total RNA input is 500ng, the proportion of 293 cell rRNA data decreased from 0.4% to 0.1%, the proportion of dahliae rRNA data decreased from 83% to 0.5%, the proportion of wheat seed rRNA data decreased from 83% to 0.7%, and the proportion of tomato fruit rRNA data decreased from 0.2% to 0.1%. At the same time, the method provided by this application is also conducive to increasing the mRNA ratio and the number of gene detections, as shown in Figures 4 and 6. When the total RNA input is 200ng, the proportion of dahliae sample mRNA data increased from 21.5% to 72.3%, and the number of gene detections increased from 2600 to 9401.

[0101] Example 2

[0102] The test was carried out using mRNA capture magnetic beads (Yeasen#12310ES96) from other companies. Except for the magnetic beads, other sample types, input amounts and experimental procedures were the same as in Example 1. The rRNA residues in the sequencing reads were counted, and the results are shown in Figures 8 and 9. When the total RNA input was 500 ng, the proportion of 293 cell rRNA data decreased from 0.9% to 0.16%, the proportion of dahlia rRNA data decreased from 96% to 0.5%, the proportion of wheat seed rRNA data decreased from 95% to 0.7%, and the proportion of tomato fruit rRNA data decreased from 0.5% to 0.1%. The results show that the method provided in this application is generally applicable to different magnetic beads, and can reduce the residual rRNA, achieving the same effect as in Example 1.

[0103] Example 3

[0104] Total RNA extracted from plant leaf samples with severe rRNA residue (raspberry, forsythia, potato, wolfberry, tea tree, bamboo, tomato, and garlic fruit), Cnidium monnieri rhizome samples, and Ganoderma lucidum rhizome samples was tested. A sample input of 500 ng was used, and the experimental process was the same as in Example 1. The rRNA residue in the sequencing reads was counted, and the results are shown in Figure 10. The results show that compared with the single-round extraction scheme of the prior art, the two-round extraction scheme provided by this application can reduce the rRNA residue in the aforementioned plant samples to over 50%.

[0105] Example 4

[0106] Based on Example 1, the two-round capture technology was further optimized, wherein only a 25°C, 5-minute incubation treatment was set in the second magnetic bead capture of mRNA step. The specific process is shown in FIG11 .

[0107] The sample and other experimental procedures were the same as those in Example 1, with two rounds of capture. The sample input was 500 ng, and the rRNA residue in the sequencing reads was counted, with the results shown in Figure 12. Comparing the data before and after optimization, the two treatment methods had little effect on rRNA residue, and the measured rRNA percentages were similar, indicating that as long as new magnetic beads were used in the second round of capture, rRNA residue could be reduced.

Claims

1. A method for mRNA enrichment, comprising the following steps: (1) mixing the total RNA sample with first capture magnetic beads and incubating, wherein the first capture magnetic beads contain Oligo(dT); (2) Applying an external magnetic field and removing the supernatant; (3) washing the first capture magnetic beads and removing the washing solution; (4) eluting the mRNA on the first capture magnetic beads to obtain an elution product; (5) applying an external magnetic field, transferring the eluted product and discarding the first capture magnetic beads, adding second capture magnetic beads to the eluted product, and incubating, wherein the second capture magnetic beads contain Oligo(dT); (6) Applying an external magnetic field and removing the supernatant; (7) washing the second capture magnetic beads and removing the washing solution; (8) Elute the mRNA from the second capture magnetic beads.

2. The method according to claim 1, wherein the incubation conditions of step (1) include incubating at 50-80°C for 1-10 minutes.

3. The method according to claim 2, wherein the incubation conditions of step (1) include first incubating at 50-80°C for 1-10 minutes and then incubating at room temperature for 1-10 minutes.

4. The method according to claim 1, wherein the incubation conditions of step (5) include incubation at room temperature for 1-10 minutes.

5. The method according to claim 4, wherein the incubation conditions of step (5) include first incubating at 50-80°C for 1-10 minutes and then incubating at room temperature for 1-10 minutes. The method according to claim 1 , wherein the washing comprises adding a washing liquid, and the washing temperature is room temperature.

7. The method according to claim 1, wherein the elution comprises adding an elution solution, and the elution condition is incubation at 50-90°C for 1-10 minutes.

8. The method of claim 1, wherein the total RNA sample is from a eukaryotic organism.

9. A method for constructing a transcriptome library, the method comprising the following steps: (1) Capturing mRNA using the method according to any one of claims 1 to 8; (2) reverse transcribing the mRNA to synthesize a first-strand cDNA; optionally, synthesizing a second-strand cDNA after the synthesis of the first-strand cDNA; (3) adding the transposase complex for incubation to obtain fragmented cDNA; (4) adding a primer with a linker sequence and obtaining an amplification product with a linker sequence by PCR amplification; (5) Optionally, purify the amplified product. 10 . The method of claim 9 , wherein the transposase complex comprises a transposase, a transposase recognition core sequence, and a tag sequence.

11. The method according to claim 9, wherein the primer further comprises an index sequence and / or a sequence complementary to the tag sequence.

12. The method of claim 9, wherein the primers include a first primer with a first adapter sequence and a second primer with a second adapter sequence.

13. The method of claim 12, wherein the first adapter sequence is a P5 adapter sequence of an Illumina platform, and the second adapter sequence is a P7 adapter sequence of an Illumina platform.

14. A method for constructing a transcriptome library, the method comprising the following steps: (1) Capturing mRNA using the method according to any one of claims 1 to 8; (2) interruption to obtain fragmented mRNA; (3) reverse transcribing the fragmented mRNA to synthesize cDNA; (4) performing end repair on the cDNA; (5) Connector connection; (6) Library amplification. The method of claim 14 , wherein the end repair comprises blunting the ends of the cDNA.

16. The method of claim 15, wherein the end repair further comprises adding adenine deoxyribonucleotide to the 3' end of the cDNA.

17. The method according to claim 14, wherein the fragmentation method is a fragmentation enzyme method, a chemical method and / or a mechanical method.

18. The method of claim 14, wherein the adapter is a Y-type adapter of the Illumina platform, a bubble adapter of the MGI platform, or a sequencing adapter of the IonTorrent platform.

19. A transcriptome analysis method, comprising the following steps: (1) constructing a sequencing library using the method described in any one of claims 9 to 18; (2) Sequencing.

20. A method for detecting mRNA expression, the method comprising: (1) Capturing mRNA using the method according to any one of claims 1 to 8; (2) Perform qRT-PCR detection.

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