A magnetic bead complex for enriching mRNA and application thereof

Abstract

The present application relates to a kind of magnetic bead complex for enriching mRNA and its application, belong to biotechnology field.The present application provides a kind of magnetic bead complex for enriching mRNA, including magnetic bead, interval arm, primer and oligodT, wherein, primer is coupled on magnetic bead by interval arm, oligodT is connected by primer with interval arm, interval arm is C chain and / or base T, primer is used to amplify target mRNA and is combined on the cDNA of magnetic bead after reverse transcription, oligodT can be specifically combined with target mRNA;The magnetic bead complex for enriching mRNA is provided with interval arm between primer and magnetic bead, on the one hand, can reduce the steric effect when in situ cDNA pre-amplification, enhance the efficiency of amplification, on the other hand, interval arm makes mRNA enrichment using the magnetic bead complex, can be in the case of magnetic bead reverse transcription, while also can carry out PCR amplification reaction.

Description

TECHNICAL FIELD

[0001] The present application relates to a magnetic bead complex for enriching mRNA and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Single-cell transcriptome sequencing (scRNA seq) is a new sequencing technology based on the second-generation high-throughput sequencing platform and bioinformatics in recent years. Compared with traditional transcriptome sequencing (bulk RNA-seq), the technical advantage is that the differences in genome, transcriptome and epigenetics between different cells can be studied at the single-cell level. Transcriptome analysis is a powerful strategy to link genotypes and cell phenotypes.

[0003] With the rapid development of molecular biology technology, the research on transcriptomics is no longer limited to the population cell level, but to the single-cell level. It is well known that the universal feature of biological tissues is cell heterogeneity. The conventional multi-cell level transcriptomics research needs a large number of cells, and the research results reflect the average value of gene expression of population cells, which masks the expression information of the small number of cell subgroups, and thus it is difficult to distinguish the transcription characteristics of different cell subgroups in population cells.

[0004] When performing cell research, the traditional bulk RNAseq usually needs hundreds of thousands to millions of cell equivalents of RNA as a template for sequencing (100 ng-5 μg). The RNA content in single cells is low, usually only 10-20 pg, which is about 10 5 ~ 10 6 The technical difficulty of the capture, amplification and library construction method is much greater than that of the traditional tissue sample RNA library construction. Therefore, the innovation and development of single-cell transcriptome sequencing technology will have great research and development value in basic research and clinical application. When performing single-cell transcriptome sequencing, obtaining high-quality mRNA is the premise of the subsequent reverse transcription amplification library construction experiment step, and is also the basis for obtaining good bioinformatics analysis results. SUMMARY

[0005] To solve the above problems, the present application provides a magnetic bead complex for enriching mRNA, which comprises magnetic beads, a spacer, a primer and oligodT; the primer is coupled to the magnetic beads through the spacer; the oligodT is connected to the spacer through the primer; the spacer is C chain and / or base T; the primer is used to amplify the cDNA of the target mRNA combined on the magnetic beads after reverse transcription; and the oligodT can specifically bind to the target mRNA.

[0006] In an embodiment of the present application, the target mRNA refers to the transcriptome of a single cell / few cells.

[0007] In an embodiment of the present application, the enrichment refers to the capture and amplification of the transcriptome of a single cell / few cells.

[0008] In an embodiment of the present application, the spacer is a carbon chain C with a length of 2-30 and / or a base T with a length of 2-30.

[0009] In an embodiment of the present application, the spacer is a carbon chain C with a length of 6-12 or a base T with a length of 4-10.

[0010] In an embodiment of the present application, the magnetic bead is a carboxyl magnetic bead.

[0011] In an embodiment of the present application, the spacer is coupled to the magnetic bead through the terminal modified NH2.

[0012] In an embodiment of the present application, the primer has a length of 12-70 bases.

[0013] In an embodiment of the present application, the primer has a length of 18-35 bases.

[0014] In an embodiment of the present application, the oligodT has a length of 12-70 bases T.

[0015] In an embodiment of the present application, the oligodT has a length of 15-35 bases T.

[0016] The present application also provides a method for preparing the magnetic bead complex for enriching mRNA, which comprises the following steps: synthesizing a spacer, a primer and an oligodT, and sequentially connecting the spacer, the primer and the oligodT to obtain a magnetic bead connecting sequence; mixing the magnetic bead and the magnetic bead connecting sequence and performing a coupling reaction to obtain the magnetic bead complex for enriching mRNA.

[0017] The present application also provides a kit for enriching mRNA, which comprises the magnetic bead complex for enriching mRNA.

[0018] In an embodiment of the present application, the kit further comprises a lysis buffer; the components of the lysis buffer comprise 1% Triton X-100 2 μL and 4 M guanidine hydrochloride / guanidinium isothiocyanate 2 μL; alternatively, the components of the lysis buffer comprise 1% Triton X-100 2 μL, 40 U / μL RNase inhibitor 0.1 μL, 2 M LiCl 1 μL, and DEPC H2O 1 μL; alternatively, the components of the lysis buffer comprise 1% Triton X-100 2 μL, 40 U / μL RNase inhibitor 0.1 μL, 2 M LiCl 0.5 μL, and DEPC H2O 1 μL; alternatively, the components of the lysis buffer comprise 1% Triton X-100 2 μL, 40 U / μL RNase inhibitor 0.1 μL, 2 M LiCl 0.5 μL, DEPC H2O 1 μL, and 1% β-mercaptoethanol 0.04 μL; alternatively, the components of the lysis buffer comprise 1% Triton X-100 2 μL, 40 U / μL RNase inhibitor 0.1 μL, 2 M LiCl 0.5 μL, DEPC H2O 1 μL, and 1% dithiothreitol 0.04 μL.

[0019] In an embodiment of the present application, the kit further comprises a hybridization buffer; the hybridization buffer is 20 mM Tris-HCl buffer, pH 7.5, added with 1.0 M LiCl, 2 mM EDTA, 5 mM DTT, and 10% formamide.

[0020] The present application also provides a single cell / few-cell transcriptome amplification and sequencing method, which comprises: using the above-mentioned magnetic bead complex for enriching mRNA or the above-mentioned kit for enriching mRNA to capture and amplify the target mRNA in a single cell / few cells, so as to realize the enrichment of the target mRNA in the single cell / few cells.

[0021] The present application also provides the application of the above-mentioned magnetic bead complex for enriching mRNA or the above-mentioned kit for enriching mRNA or the above-mentioned single cell / few-cell transcriptome amplification and sequencing method in single cell / few-cell transcriptome amplification and sequencing.

[0022] The technical scheme of the present application has the following advantages:

[0023] 1. The application provides a magnetic bead complex for enriching mRNA, the magnetic bead complex for enriching mRNA comprises magnetic beads, an arm, a primer and oligodT, wherein the primer is coupled to the magnetic beads through the arm, the oligodT is connected to the arm through the primer, the arm is C chain and / or base T, the primer is used for amplifying the cDNA of the target mRNA combined on the magnetic beads after reverse transcription, and the oligodT can be specifically combined with the target mRNA; compared with the traditional oligodT magnetic beads, the magnetic bead complex for enriching mRNA sets the arm between the primer and the magnetic beads, on the one hand, the steric hindrance effect during the in-situ cDNA pre-amplification can be reduced, and the amplification efficiency is enhanced, on the other hand, the arm enables the mRNA enrichment using the magnetic bead complex to be carried out in the presence of the magnetic beads, and the reverse transcription and the PCR amplification reaction can be carried out at the same time, and by connecting the arm, the polyA is more easily combined with the magnetic beads.

[0024] Further, the magnetic bead complex for enriching mRNA can be used for enriching single cell / low cell mRNA, and is particularly effective for samples (with interfering substances and a large amount of rRNA) which are not good for direct lysis, the obtained mRNA can be subjected to single cell and low cell transcriptome NGS sequencing, and can also be used for detection of the expression level of special genes; since the cDNA synthesized by reverse transcription is attached to the magnetic beads, the PCR amplification reaction can be repeatedly carried out, so that the cDNA product of the sample can be saved for later research.

[0025] Further, since the single cell / low cell contains a relatively small amount of mRNA, after the mRNA is captured using the magnetic bead complex for enriching mRNA, the operation of elution can be omitted, and the reverse transcription and PCR amplification can be directly carried out, so that the loss of mRNA in the sample is reduced, and during the PCR reaction, the primer is more easily combined with the template, so that the efficiency of PCR amplification is ensured.

[0026] 2. The application provides a kit for enriching mRNA, the kit comprises a magnetic bead complex for enriching mRNA, a lysis buffer and a hybridization buffer, the kit optimizes the cell lysis cell liquid formula, increases the amount of RNA released by cell lysis, and optimizes the hybridization buffer formula, increases the ability of the magnetic beads to capture RNA. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Structure diagram of the magnetic bead complex for enriching mRNA.

[0028] Figure 2 Transcriptome amplification result quality control chart obtained by using different lysis liquid formulas. Figure 2In the figure, A is the quality control chart of the transcriptome amplification results obtained using lysate A, B is the quality control chart of the transcriptome amplification results obtained using lysate B, C is the quality control chart of the transcriptome amplification results obtained using lysate C, D is the quality control chart of the transcriptome amplification results obtained using lysate D, and E is the quality control chart of the transcriptome amplification results obtained using lysate E.

[0029] Figure 3 : Quality control chart of the transcriptome amplification results obtained using different magnetic beads. Figure 3 In the figure, A and B are the quality control charts of the transcriptome amplification results obtained using the magnetic bead complex of Comparative Example 1 (A and B are duplicate samples), and C and D are the quality control charts of the transcriptome amplification results obtained using the magnetic bead complex of Example 1 (C and D are duplicate samples).

[0030] Figure 4 : Number of genes detected by sequencing of the transcriptome obtained using different magnetic beads. Figure 4 In the figure, A and B are the number of genes detected by sequencing of the transcriptome obtained using the magnetic bead complex of Comparative Example 1 (A and B are duplicate samples), and C and D are the number of genes detected by sequencing of the transcriptome obtained using the magnetic bead complex of Example 1 (C and D are duplicate samples).

[0031] Figure 5 : Quality control chart of the transcriptome amplification results obtained using different magnetic beads. Figure 5 In the figure, WD1 is the quality control chart of the transcriptome amplification results obtained using the magnetic beads of Qiagen, and N1 is the quality control chart of the transcriptome amplification results obtained using the magnetic bead complex of Example 1.

[0032] Figure 6 : Electrophoretic chart of the transcriptome amplification results obtained using different magnetic beads. Figure 6 In the figure, WD1 and WD2 are the electrophoretic charts of the transcriptome amplification results obtained using the magnetic beads of Qiagen (WD1 and WD2 are duplicate samples), and N1 and N2 are the electrophoretic charts of the transcriptome amplification results obtained using the magnetic bead complex of Example 1 (N1 and N2 are duplicate samples).

[0033] Figure 7 : Quality control chart of the transcriptome amplification results obtained using different methods. Figure 7 In the figure, G is the quality control chart of the transcriptome amplification results obtained using the coupled magnetic beads method of Example 7, and E is the quality control chart of the transcriptome amplification results obtained using the Smart-seq2 method.

[0034] Figure 8 : Number of genes detected by sequencing of the transcriptome obtained using different methods. Figure 8 In the figure, G is the number of genes detected by sequencing of the transcriptome obtained using the coupled magnetic beads method of Example 7, and E is the number of genes detected by sequencing of the transcriptome obtained using the Smart-seq2 method.

[0035] Figure 9 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer C2.

[0036] Figure 10 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer C6.

[0037] Figure 11 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer C12.

[0038] Figure 12 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer T6.

[0039] Figure 13 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer C6T6.

[0040] Figure 14 : Amplification result quality control plot of the transcriptome obtained using magnetic bead complex with spacer C12T6. DETAILED DESCRIPTION

[0041] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application, which is defined by the appended claims. Any product that is the same as or similar to the present application that is derived from the present application or from the combination of the present application with other prior art features, falls within the scope of the present application.

[0042] In the following examples, the specific experimental procedures or conditions are not specified, and can be performed according to the conventional experimental procedures described in the literature in the art. The reagents or instruments used are not specified, and are all conventional reagents that can be obtained commercially.

[0043] The materials involved in the following examples are as follows:

[0044] 1% Carboxyl magnetic beads suspension (COOH Sera-Mag Carboxylate-Modified Magnetic Particles): purchased from GE, particle size 1 μm, 1% means 10 mg of magnetic bead particles in 1 mL of solution;

[0045] 10% EDAC: 10 mg of EDAC (carbodiimide) was dissolved in 1 mL of ultrapure water to obtain 10% EDAC, which was prepared and used immediately.

[0046] Example 1: Magnetic bead complex for enriching mRNA and preparation thereof

[0047] As Figure 1As shown, the present embodiment provides a magnetic bead complex for enriching mRNA, which is composed of carboxyl magnetic beads, a C6 spacer, a primer with a nucleotide sequence of SEQ ID NO. 1, and an oligodT with a nucleotide sequence of SEQ ID NO. 2 and modified with NH2 at the end (i.e. NH2-[C6]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN); the spacer is coupled to the magnetic beads through the end-modified NH2; the primer is coupled to the magnetic beads through the spacer; the oligodT is connected to the spacer through the primer; the primer is used to amplify the cDNA of the target mRNA after reverse transcription and combined on the magnetic beads; and the oligodT can specifically bind to the target mRNA.

[0048] The preparation method of the magnetic bead complex for enriching mRNA includes the following steps:

[0049] 1. Synthesize the spacer, primer, and oligodT, and sequentially connect the spacer, primer, and oligodT to obtain a magnetic bead connection sequence (the synthesis and connection of the spacer, primer, and oligodT are completed by Beijing Ruibo Xingke Biotechnology Co., Ltd.);

[0050] 2. Vortex 1% carboxyl magnetic bead suspension with a vortex instrument to obtain vortexed carboxyl magnetic bead suspension;

[0051] 3. Mix 160 μL of DNase / RNase-free water, 500 μL of 0.1M MES coupling buffer, 200 μL of vortexed carboxyl magnetic bead suspension, 40 μL of 10 μM magnetic bead connection sequence, and 100 μL of 10% EDAC, and react at 37°C and 200 rpm for 12h to obtain a reaction solution; centrifuge the reaction solution at 1000 rpm for 5 min, discard the supernatant, and take the precipitate;

[0052] 4. Wash the precipitate with the same volume of RNase-free water as the reaction solution twice, then wash with the same volume of 0.1M imidazole aqueous solution at pH 6.0 as the reaction solution twice (each washing requires shaking at 37°C and 200 rpm for 5 min), then wash with the same volume of 0.1M sodium bicarbonate aqueous solution as the reaction solution three times (each washing requires shaking at 37°C and 200 rpm for 5 min), and finally wash with the same volume of 0.1M sodium bicarbonate aqueous solution as the reaction solution twice (each washing requires shaking at 65°C and 200 rpm for 30 min) to obtain the magnetic bead complex for enriching mRNA.

[0053] Example 2: Kit for enriching mRNA in a sample

[0054] The present embodiment provides a kit for enriching mRNA in a sample, which is composed of the magnetic bead complex for enriching mRNA of embodiment 1, a lysis buffer A (formula see Table 1), and a hybridization buffer; the hybridization buffer is a 20 mM, pH 7.5 Tris-HCl buffer added with 1.0 M LiCl (lithium chloride), 2 mM EDTA (ethylenediaminetetraacetic acid), 5 mM DTT (dithiothreitol), and 10% (10% refers to mass volume concentration, i.e. g / 100 mL) formamide.

[0055] Table 1 Formula of lysis buffer A

[0056] Component Volume (μL) (1 x) Notes 1% Triton X-100 2 1% refers to mass by volume concentration, i.e. g / 100 mL GuHCl / Guanidine isothiocyanate 4M 2 DEPC H2O 0

[0057] Embodiment 3: Kit for enriching mRNA in a sample

[0058] The present embodiment provides a kit for enriching mRNA in a sample, which uses a lysis buffer B (formula see Table 2) compared with embodiment 2.

[0059] Table 2 Formula of lysis buffer B

[0060]

[0061]

[0062] Embodiment 4: Kit for enriching mRNA in a sample

[0063] The present embodiment provides a kit for enriching mRNA in a sample, which uses a lysis buffer C (formula see Table 3) compared with embodiment 2.

[0064] Table 3 Formula of lysis buffer C

[0065] Component Volume (μL) (1 x) Notes 1% Triton X-100 2 1% refers to mass by volume concentration, i.e. g / 100 mL RNase inhibitor (40 U / μL) 0.1 LiCl 2M 0.5 DEPC H2O 1

[0066] Embodiment 5: Kit for enriching mRNA in a sample

[0067] The present embodiment provides a kit for enriching mRNA in a sample, which uses a lysis buffer D (formula see Table 4) compared with embodiment 2.

[0068] Table 4 Formula of lysis buffer D

[0069] Component Volume (μL) (1 x) Notes 1% Triton X-100 2 1% refers to mass by volume concentration, i.e. g / 100 mL RNase inhibitor (40 U / μL) 0.1 LiCl 2M 0.5 DEPC H2O 1 1% BME (β mercaptoethanol) 0.04 1% refers to mass by volume concentration, i.e. g / 100 mL

[0070] Embodiment 6: Kit for enriching mRNA in a sample

[0071] The present embodiment provides a kit for enriching mRNA in a sample, which uses lysis buffer E (formula see Table 4) compared with embodiment 2.

[0072] Table 5 Formula of lysis buffer D

[0073]

[0074]

[0075] Embodiments 7-11: A single cell / small number of cells transcriptome amplification and sequencing method

[0076] The present embodiment provides a single cell / small number of cells transcriptome amplification and sequencing method, which uses the kits for enriching mRNA in a sample of embodiments 2-6 respectively, comprising the following steps:

[0077] mRNA capture step: pick up single cells (tumor cell lines H-1975, SW620, purchased from Beijing Union Cell Resource Center) by micromanipulation; put the picked single cells into a PCR tube containing 4 μL cell lysis solution; after adding 1 μL magnetic bead complex for enriching mRNA into the PCR tube, place it at room temperature (25°C) for 2 min, vortex for 20 s with a vortex instrument, incubate at 72°C for 3 min, quickly place it on ice, and place it at room temperature (25°C) for 5 min to obtain a cell lysis system; after adding 16 μL hybridization buffer into the PCR tube, place it at room temperature (25°C) for 5 min to obtain a hybridization system; centrifuge the PCR tube at 1000 rpm for 5 min, and discard the supernatant; wash the precipitate in the PCR tube with 40 μL 6×SSC once, and centrifuge it at 1000 rpm for 5 min, and discard the supernatant;

[0078] Reverse transcription step: mix reverse transcription component A (ingredients see Table 6) and reverse transcription component B (ingredients see Table 7) with the magnetic beads obtained in the mRNA capture step in the PCR tube (the total volume after mixing is 10 μL), and react at 42°C for 90 min, shake once every 10 min during the reaction process, to obtain a reverse transcription system; mix the reverse transcription system and 50 μL TE-SDS in the PCR tube, inactivate the RT enzyme, centrifuge at 1000 rpm for 5 min, and discard the supernatant; wash the precipitate in the PCR tube with 50 μL TE-TW and 50 μL 10 mM, pH 8.0 Tris-HCL once, and centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0079] Enzyme digestion step: mix the enzyme digestion reaction mixture (ingredients see Table 8) with the magnetic beads obtained in the reverse transcription step in a PCR tube, react at 37°C for 60 min, shake every 10 min during the reaction process, obtain the digestion system; centrifuge the PCR tube at 1000 rpm for 5 min, discard the supernatant; wash the precipitate in the PCR tube with 50 μL TE-TW and 50 μL 10 mM, pH 8.0 Tris-HCL respectively, then centrifuge at 1000 rpm for 5 min, discard the supernatant;

[0080] Pre-amplification step: mix the PCR reaction mix (ingredients see Table 9) with the magnetic beads obtained in the Exo I enzyme digestion step in a PCR tube, then perform PCR reaction (PCR reaction program see Table 10), obtain the PCR amplification product; use the full-automatic biological fragment analyzer Qsep400 to perform quality inspection on the PCR product, determine the size of the DNA fragment, and the quality inspection results of the kits for enriching mRNA in samples obtained in Examples 2-6 are shown in Figure 2 ;

[0081] Library construction and sequencing step: use the Nuoyuan kit (TruePrep DNA Library Prep Kit V2 for Illumina, item number TD501-TD503) to construct a transcriptome library for the PCR amplification product obtained in the pre-amplification step that passes the quality inspection; after library quality inspection, use the illumina sequencing platform for sequencing, the sequencing strategy is PE150; after the sequencing data is downloaded, use single cell transcriptome analysis software for bioinformatics analysis.

[0082] It can be known from Figure 2 that the kits for enriching mRNA in samples of Examples 2-6 can all realize transcriptome amplification and sequencing of single cells / low amount of cells, among which the kits for enriching mRNA in samples of Examples 5-6 (i.e. using cell lysis solution D-E) have better effect on transcriptome amplification and sequencing of single cells / low amount of cells.

[0083] Table 6 Reverse transcription component A

[0084] component volume(μL) dNTP (10 μM) 1 RNase inhibitor (40 U / μL) 0.1 ddH2O 3.49

[0085] Table 7 Reverse transcription component B

[0086] component volume(μL) first strand buffer 2 TSO (100 μM) 0.1 RNase inhibitor (40 U / μL) 0.25 RTase 0.5 DTT (0.1 M) 0.5 Betaine (5 M) 2 MgCl2(1M) 0.06

[0087] Table 8 Enzyme digestion reaction mixture

[0088]

[0089]

[0090] Table 9 PCR reaction mix

[0091]

[0092] Table 10 PCR reaction procedure

[0093]

[0094] Comparative Example 1: Magnetic bead complexes for mRNA enrichment and their preparation

[0095] This comparative example provides a magnetic bead complex for enriching mRNA, wherein the C6 intercostal arm has been removed compared to Example 1.

[0096] Comparative Example 2: Kit for enriching mRNA in samples

[0097] This comparative example provides a kit for enriching mRNA in a sample, which, compared to Example 2, uses the magnetic bead complex for enriching mRNA from Comparative Example 1.

[0098] Comparative Example 3: A method for single-cell / small-cell transcriptome amplification and sequencing

[0099] This comparative example provides a method for single-cell / small-cell transcriptome amplification and sequencing, which, based on Example 11, uses the kit for enriching mRNA in samples from Comparative Example 2. The quality control results obtained using the kit for enriching mRNA in samples from Comparative Example 2 are shown below. Figure 3 (Compared to Example 11). Bioinformatics analysis results of sequencing data obtained using the kit for enriching mRNA in samples (Comparative Example 2) are shown below. Figure 4 And Table 11 (with Example 11 as a comparison).

[0100] Depend on Figure 3 It can be seen that, compared with the kit for enriching mRNA in samples in Comparative Example 2 (i.e., using a magnetic bead complex without interdigitated arms), the kit for enriching mRNA in samples in Example 6 (i.e., using a magnetic bead complex with interdigitated arms) yielded longer fragments and better results in single-cell / small-cell transcriptome amplification. Figure 4 As shown in Table 11, there was no significant difference in sequencing quality and alignment rate between single-cell / small-cell transcriptome sequencing using the kits for enriching mRNA in samples from Example 6 and Comparative Example 2. However, compared with the kit for enriching mRNA in samples from Comparative Example 2 (i.e., using magnetic bead complexes without interstitial arms), the kit for enriching mRNA in samples from Example 6 (i.e., using magnetic bead complexes with interstitial arms) detected more genes and achieved better results in single-cell / small-cell transcriptome sequencing.

[0101] Table 11. Transcriptome sequencing data quality and alignment rate

[0102] Sample Q20％ MapRate (Map Rate) A 95.68 87.20％ B 92.33 92.82％ C 96.42 88.23％ D 97.58 91.32％

[0103] In Table 11, A and B represent the transcriptome sequencing data quality and alignment rate of transcriptomes obtained using the magnetic bead complex of Comparative Example 1 (A and B are duplicate samples), and C and D represent the transcriptome sequencing data quality and alignment rate of transcriptomes obtained using the magnetic bead complex of Example 1 (C and D are duplicate samples).

[0104] Comparative Example 4: Kit for enriching mRNA in samples

[0105] This comparative example provides a kit for enriching mRNA in samples. Compared with Example 2, the kit uses oligodT magnetic beads (model MS04T) purchased from Suzhou Weidu Biotechnology Co., Ltd.

[0106] Comparative Example 5: A method for single-cell / small-cell transcriptome amplification and sequencing

[0107] This comparative example provides a method for single-cell / small-cell transcriptome amplification and sequencing, which, based on Example 11, uses the kit for enriching mRNA in samples from Comparative Example 4. The quality control results obtained using the kit for enriching mRNA in samples from Comparative Example 4 are shown below. Figure 5 (Using Example 11 as a control). Using Example 11 as a control, the quality-controlled PCR amplification products obtained using the kit for enriching mRNA in samples (Comparative Example 4) were analyzed by gel electrophoresis. The results are shown in [Figure 11]. Figure 6 .

[0108] Depend on Figure 5-6 It can be seen that, compared with the kit for enriching mRNA in samples in Comparative Example 4 (i.e., using ordinary oligodT magnetic beads), the kit for enriching mRNA in samples in Example 6 (i.e., using magnetic bead complex) for single-cell / small-cell transcriptome amplification yields a larger proportion of large cDNA fragments and higher quality.

[0109] Comparative Example 6: A method for single-cell / small-cell transcriptome amplification and sequencing

[0110] This comparative example provides a method for single-cell / small-cell transcriptome amplification and sequencing, which is the Smart-seq2 method, including the following steps:

[0111] Cell lysis step: pick up single cells (tumor cell lines H-1975, SW620, purchased from Beijing Union Cell Resource Center) by micromanipulation; add the picked single cells to 4 μL cell lysis solution to obtain a mixed solution; vortex the mixed solution for 20 s, incubate at 72 °C for 3 min, quickly place on ice, and place at room temperature (25 °C) for 5 min to obtain a cell lysis system;

[0112] Reverse transcription step: after mixing the reaction mixture (ingredients see Table 12) with the cell lysis system obtained in the cell lysis step, react at 25 °C for 5 min, at 42 °C for 60 min, at 50 °C for 30 min, at 72 °C for 10 min, and at 4 °C to obtain a reverse transcription system;

[0113] Pre-amplification step: after mixing the PCR reaction mix (ingredients see Table 9) with the reverse transcription system obtained in the reverse transcription step, perform PCR reaction (PCR reaction program see Table 10) to obtain a PCR amplification product; after purifying the PCR product using XP magnetic beads twice, elute with 20 μL NF-H2O to obtain the purified PCR product; use the full-automatic biological fragment analyzer Qsep400 to perform quality inspection on the purified PCR product to determine the size of the DNA fragment, and the quality inspection result is shown in Figure 7 (Example 11 as a control);

[0114] Library construction and sequencing step: use the Nuoyuan kit (TruePrep DNA Library Prep Kit V2 for Illumina, item number TD501-TD503) to construct a transcriptome library from the qualified PCR amplification product obtained in the pre-amplification step; after library quality inspection, use the illumina sequencing platform for sequencing, and the sequencing strategy is PE150; after sequencing data is downloaded, use single cell transcriptome analysis software for bioinformatics analysis, and the analysis result is shown in Figure 8 (Example 11 as a control).

[0115] It can be seen from Figure 7 that compared with the Smart-seq2 method, the proportion of large fragment cDNA of the amplification product obtained by using the magnetic bead coupling method of Example 6 for single cell / low amount of cell transcriptome amplification is more, mainly concentrated at about 2000 bp, and the effect is better. From Figure 8 it can be seen that compared with the Smart-seq2 method, the number of genes detected by using the magnetic bead coupling method of Example 6 for single cell / low amount of cell transcriptome sequencing is more, and the effect is better.

[0116] Table 12 Reaction mixture

[0117] component volume(μL) SuperScript II reverse transcriptase (200 U / μL) 0.5 RNAse inhibitor (40 U / μL) 0.25 Superscript II first-strand buffer (5x) 2 0.1 M DTT 0.5 Betaine (5 M) 2 MgCl2 (1 M) 0.06 TSO (100 uM) 0.1 Nuclease-free H2O 0.29

[0118] Examples 12-16: Magnetic bead complexes for mRNA enrichment and their preparation

[0119] This embodiment provides a magnetic bead complex for enriching mRNA. Compared with Example 1, the intercostal arms of the magnetic bead complex for enriching mRNA are C2 (i.e., NH2-[C2]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN), C12 (i.e., NH2-[C12]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN), T6 (i.e., NH2-[T6]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN), C6T6 (i.e., NH2-[C6T6]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN), or C12T6 (i.e., NH2-[C12T6]-AAGCAGTGGTATCAACGCAGAGTAC-[T30]VN).

[0120] Examples 17-21: Kits for Enriching mRNA in Samples

[0121] This embodiment provides a kit for enriching mRNA in a sample, which, compared with Example 11, uses the magnetic bead complexes of Examples 12-16.

[0122] Examples 22-26: A method for single-cell / small-cell transcriptome amplification and sequencing

[0123] This embodiment provides a method for single-cell / small-cell transcriptome amplification and sequencing. The method is based on Example 11, using the kits for enriching mRNA in samples from Examples 17-21. The quality control results obtained using the kits for enriching mRNA in samples from Examples 17-21 are shown below. Figure 9-14 (in, Figure 10 (To repeat the results obtained in Example 11).

[0124] Depend on Figure 9-14 It can be seen that the main peak length of the amplified product fragments obtained by single-cell / small-cell transcriptome amplification using the kits for enriching mRNA in samples (i.e., using magnetic bead complexes containing different lengths of intercellular arms) in Examples 1, 12-16 is all above 1000kb. Among them, the amplified product fragments obtained by single-cell / small-cell transcriptome amplification using the kit for enriching mRNA in samples (i.e., using magnetic bead complexes with intercellular arm sequence C12T6) in Example 16 have a higher proportion of large cDNA fragments and higher quality.

[0125] Obviously, the above embodiments are merely exemplary and not intended to limit the embodiments. Based on the above description, one of ordinary skill in the art can make other variations and changes of different forms. Here, it is not necessary and impossible to enumerate all the embodiments. The obvious variations and changes derived therefrom are still within the protection scope of the present application. SEQUENCE LISTING <110> Suzhou Geekgene Biotech Co., Ltd. <120> A magnetic bead complex for enriching mRNA and application thereof <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <400> 1 aagcagtggt atcaacgcag agtac 25 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (32)..(32) <223> n is a, c, g, or t <400> 2 tttttttttt tttttttttt tttttttttt vn 32

Claims

1. A magnetic bead complex for enriching mRNA, characterized in that, The magnetic bead complex for enriching mRNA includes magnetic beads, a septal arm, a primer, and oligodT; the primer is coupled to the magnetic bead via the septal arm; and oligodT is linked to the septal arm via the primer. The intercalary arm is a C-chain and / or a T-base; the primer is used to amplify the cDNA bound to the magnetic bead after reverse transcription of the target mRNA; the oligodT can specifically bind to the target mRNA; the intercalary arm is a 6-12 carbon chain C and / or a 4-10 base T.

2. The magnetic bead complex for enriching mRNA as described in claim 1, characterized in that, The primers are 12 to 70 bases in length.

3. The magnetic bead complex for enriching mRNA as described in claim 1 or 2, characterized in that, The length of oligodT is 12 to 70 base T.

4. A method for preparing the magnetic bead complex for enriching mRNA as described in any one of claims 1 to 3, characterized in that, The method is as follows: synthesizing a septal arm, a primer, and oligodT, and sequentially linking the septal arm, primer, and oligodT to obtain a magnetic bead linker sequence; mixing the magnetic beads and the magnetic bead linker sequence and performing a coupling reaction to obtain the magnetic bead complex for enriching mRNA as described in any one of claims 1 to 3.

5. A kit for enriching mRNA, characterized in that, The kit comprises a magnetic bead complex for enriching mRNA as described in any one of claims 1 to 3.

6. The kit for enriching mRNA as described in claim 5, characterized in that, The kit also includes a lysis buffer; the lysis buffer comprises 2 μL of 1% Triton X-100 and 2 μL of 4M guanidine hydrochloride / guanidine isothiocyanate; or, the lysis buffer comprises 2 μL of 1% Triton X-100, 0.1 μL of 40 U / μL RNase inhibitor, 1 μL of 2M LiCl, and 1 μL of DEPC H2O; or, the lysis buffer comprises 2 μL of 1% Triton X-100, 0.1 μL of 40 U / μL RNase inhibitor, 0.5 μL of 2M LiCl, and 1 μL of DEPC H2O; or, the lysis buffer comprises 2 μL of 1% Triton X-100, 0.1 μL of 40 U / μL RNase inhibitor, 0.5 μL of 2M LiCl, 1 μL of DEPCH2O, and 0.04 μL of 1% β-mercaptoethanol; or, the lysis buffer comprises 1% Triton X-100... X-100 2μL, 40U / μL RNase inhibitor 0.1μL, 2M LiCl 0.5μL, DEPC H2O 1μL and 1% dithiothreitol 0.04μL.

7. The kit for enriching mRNA as described in claim 5 or 6, characterized in that, The kit also includes a hybridization buffer; the hybridization buffer is a 20mM Tris-HCl buffer at pH 7.5 containing 1.0M LiCl, 2mM EDTA, 5mM DTT and 10% formamide.

8. A method for single-cell / small-cell transcriptome amplification and sequencing, characterized in that, The method includes: capturing and amplifying target mRNA in single cells / a small number of cells using the magnetic bead complex for mRNA enrichment as described in any one of claims 1 to 3 or the kit for mRNA enrichment as described in any one of claims 5 to 7.

9. The application of the magnetic bead complex for enriching mRNA as described in any one of claims 1 to 3, the kit for enriching mRNA as described in any one of claims 5 to 7, or the single-cell / small-cell transcriptome amplification and sequencing method as described in claim 8 in single-cell / small-cell transcriptome amplification and sequencing.

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