Preparation method of efficient and high-throughput plant tissue mRNA-seq sequencing library

Abstract

The invention discloses a preparation method of a high-efficiency and high-throughput plant tissue mRNA-seq sequencing library. The preparation method comprises the following steps: grinding a plant sample and Trizol; 1-bromo-3-chloropropane is added into the reaction kettle; taking supernatant, and adding isopropanol to precipitate RNA; centrifuging, and discarding supernatant to obtain white precipitate; rinsing and precipitating; discarding redundant liquid, airing the precipitate to a semitransparent state, and dissolving the precipitate; enriching mRNA (messenger ribonucleic acid); carrying out cDNA one-chain synthesis; carrying out Tn5 enzyme digestion; purifying an enzyme digestion product; carrying out PCR library amplification; purifying and sorting PCR products; and collecting the library supernatant into a new PCR plate. Compared with the traditional mRNA-seq, the mRNA-seq library has the advantages that the synthesis of a cDNA second chain, the addition of terminal repair A and the additional connection of a sequencing joint are reduced, the operation process is greatly simplified, and the mRNA-seq library of 96 different tissue samples can be constructed within one day.

Description

Technical Field

[0001] The present invention relates to a method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library. Background Art

[0002] Transcriptome refers to the characteristics and quantity of a complete set of transcripts in a cell during a specific growth period or under specific physiological conditions. Transcriptomics research mainly includes transcript abundance, classification, gene transcription structure and post-transcriptional modification, aiming to reveal the mechanism by which transcripts participate in regulating genome structure and complex biological processes.

[0003] RNA-seq is a powerful tool for transcriptomics research. It can quickly obtain cell or tissue-specific transcript nucleic acid sequences, gene expression abundance, transcript structure, and alternative splicing types through deep sequencing. The main experimental steps of traditional RNA-seq include extraction of total RNA from cells or tissues, mRNA capture or rRNA removal, RNA fragmentation, cDNA one-strand synthesis, two-strand synthesis, end repair and A addition, sequencing adapter connection, and library amplification. The process is relatively cumbersome and costly. Tn5 transposase is widely used in the construction of second-generation sequencing DNA libraries. It can randomly mark and cut double-stranded DNA and double-stranded RNA:DNA, and has been proven to be used for the construction of RNA-seq libraries. The representative technologies are SHERRY (Sequencing HEteRo RNA-DNA-hYbrid) and Smart-seq2, and the test objects are mainly animal cells. The above methods can usually only be applied to animal experiments with a small number of samples, and cannot achieve the preparation of large-scale RNA-seq libraries in a short period of time, especially in plant transcriptome research. Therefore, it is necessary to develop a method for preparing a high-throughput transcriptome library for plants. Based on the above technology (SHERRY), this technology simplifies the operation steps, reduces the experimental cost, and realizes the preparation of mRNA-seq libraries of at least 96 different plant tissue samples within one day. Summary of the invention

[0004] In order to solve the defects of the prior art, the present invention provides a method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library, comprising the following steps:

[0007] (1) Grind the plant sample and Trizol together, let stand at room temperature, and then centrifuge at room temperature;

[0008] (2) adding 1-bromo-3-chloropropane to the ground sample in step (1), vortexing, standing at room temperature, and then centrifuging at 4° C.;

[0009] (3) taking the supernatant after centrifugation in step (2), adding isopropanol, inverting to mix, placing at -20°C for alcohol precipitation, and then centrifuging at 4°C;

[0010] (4) Discard the supernatant after centrifugation in step (3), and a white precipitate will be visible; rinse the precipitate with 75% ethanol and then centrifuge at 4°C;

[0011] (5) discarding the supernatant after centrifugation in step (4), and then centrifuging at 4°C;

[0012] (6) discarding the excess liquid after centrifugation in step (5), drying the precipitate until it becomes translucent, and then dissolving the precipitate with RNase-free water;

[0013] (7) Measure the total RNA concentration using NanoDrop2000; Take the total RNA into the PCR plate and use vazyme mRNAcapture beads to enrich mRNA from the total RNA;

[0014] (8) cDNA single-strand synthesis;

[0015] (9) performing Tn5 digestion;

[0016] (10) Add DNA Clean beads, pipette to mix, and let stand at room temperature;

[0017] (11) Discard the supernatant on the magnetic stand and add 80% ethanol to rinse the beads. Repeat twice.

[0018] (12) Discard excess ethanol and dry the beads; elute with RNase-free water; collect the supernatant into a new PCR plate;

[0019] (13) PCR library amplification;

[0020] (14) Add 0.55× DNA Clean beads to the PCR product obtained in step (13), pipette and mix, and let stand at room temperature; then collect the supernatant on a magnetic rack and place it in a new PCR plate;

[0021] (15) Add 0.25× DNA Clean beads to the supernatant collected in step (14), pipette and mix, and let stand at room temperature; then discard the supernatant on the magnetic rack, add 80% ethanol to rinse the beads, and repeat twice;

[0022] (16) Discard excess ethanol and dry the beads; elute with RNase-free water; collect the library supernatant into a new PCR plate;

[0023] (17) Detect the concentration of mRNA-seq library.

[0024] Preferably, in step (1), a ball mill is used for grinding, the ball mill is quick-frozen with liquid nitrogen, a 96-well deep-well plate is used to place the material to be ground, and the material is allowed to stand at room temperature for 5 to 10 minutes; the centrifugal speed is 4000 to 5000 rpm, and the centrifugal time is 3 to 6 minutes.

[0025] Preferably, in step (2), the mixture is vortexed for 30 to 45 seconds and allowed to stand at room temperature for 10 to 18 minutes; the centrifugal speed is 4000 to 5000 rpm, and the centrifugal time is 15 to 20 minutes.

[0026] Preferably, the volume ratio of the supernatant after centrifugation and isopropanol taken in step (3) is 1:1, and the alcohol precipitation time is not less than 30 minutes; the centrifugal speed is 4000-5000 rpm, and the centrifugal time is 20-25 minutes; the centrifugal speed in step (4) is 4000-5000 rpm, and the centrifugal time is 5-10 minutes; the centrifugal speed in step (5) is 4000-5000 rpm, and the centrifugal time is 5-10 minutes.

[0027] Preferably, the step (6) is dissolved for 10 to 20 minutes, and the 96-well deep-well plate is placed in a metal bath at 60 to 70° C. for dissolution.

[0028] Preferably, the reaction system in each well in step (8) is as follows: 10 μL mRNA (10 ng-100 ng), 2 μL oligod (T) 23 VN Primer (50μM), 2μL 10×M-MuLV buffer, 1μL M-MuLV RT (200U / μL), 1μL 10mM dNTPs mix, 0.5μL RNase Inhibitor (40U / μL), 3.5μL RNase-free Water; place the reaction system on the PCR instrument and react according to the following program: Lid, 105℃; 42℃, 1h; 65℃, 20min.

[0029] Preferably, the following reaction system is used for Tn5 digestion in step (9): 4 μL TTEmixV50, 8 μL 5×TTBL, 20 μL of the reaction product in step (9), 8 μL RNase-free Water; the above system is placed on a PCR instrument and reacted according to the following procedure: 105° C., Lid; 55° C., 10 min; 4° C., Hold.

[0030] Preferably, the step (13) uses the following reaction system for PCR library amplification: 10 μL 5×TAB, 5 μL PPM, 1 μL TAE, 5 μL N6XX, 5 μL N8XX, 24 μL supernatant in step 21; the above system is placed on a PCR instrument and reacted according to the following program: 105°C, Lid; 72°C, 3 min; 98°C, 3 min; 98°C, 15 s, 60°C, 30 s, 72°C, 1 min, the above temperatures are performed for 8-11 cys; 72°C, 5 min; 4°C, Hold.

[0031] Preferably, the step (10) is allowed to stand at room temperature for 5 to 10 minutes; and the step (14) is allowed to stand at room temperature for 5 to 10 minutes.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The entire operation of the present invention is carried out in a 96-deep-well plate and a 96-well PCR plate, and the mRNA-seq library construction of no less than 96 different tissue samples can be realized simultaneously.

[0034] The present invention includes total RNA extraction, mRNA enrichment, cDNA single-strand synthesis, Tn5 transposition, PCR amplification, and library fragment screening. Compared with traditional mRNA-seq, it lacks the synthesis of the second strand of cDNA, end repair and A addition, and the additional connection of sequencing adapters, which greatly simplifies the operation process. The mRNA-seq library of 96 different tissue samples can be constructed within one day.

[0035] The present invention uses a ball grinder to wet grind and lyse to obtain Total RNA from plant tissues. This method greatly improves the efficiency of Total RNA extraction. Compared with the requirement of not less than 0.1g of plant tissue sample required for dry grinding in a mortar in the traditional Total RNA extraction method, the present invention only requires 0.01g-0.05g of tissue sample to extract high-quality and high-concentration Total RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a flow chart of the HRNA-seq experiment of the present invention;

[0037] Figure 2 It is a schematic diagram comparing the HRNA-seq signal and the Truseq RNA-seq signal obtained in Example 1 of the present invention;

[0038] Figure 3 It is a schematic diagram comparing the number of genes detected by HRNA-seq and Truseq RNA-seq in Example 1 of the present invention;

[0039] Figure 4 It is a schematic diagram comparing the technical repeatability of HRNA-seq and Truseq RNA-seq in Example 1 of the present invention;

[0040] Figure 5 It is a schematic diagram of the distribution of HRNA-seq and Truseq RNA-seq signals on genes in Example 1 of the present invention;

[0041] Figure 6 It is a schematic diagram comparing the GC content of HRNA-seq and Truseq RNA-seq libraries in Example 1 of the present invention;

[0042] Figure 7 It is a schematic diagram of the effect of HRNA-seq and Truseq RNA-seq methods on the relative expression of genes in Example 1 of the present invention;

[0043] Figure 8 It is the library sequencing map of HRNA-seq.Rep1 and HRNA-seq.Rep2 in Example 1 of the present invention.

[0044] The Trueseq RNA-seq data mentioned in this article were downloaded from the RiceENCODE database with SRA numbers SRR10751892 and SRR10751893. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0046] Example 1: Preparation of an efficient and high-throughput mRNA-seq sequencing library.

[0047] (1) Add 3 mm grinding steel beads to a 96-well deep-well plate, 3 beads per well; then add about 0.01-0.05 g of the corresponding rice MH63 15-day-old seedling fresh sample to each well; during this operation, the sample needs to be placed in liquid nitrogen throughout the process;

[0048] (2) Add 1 ml of Trizol to each well of the 96-well deep-well plate prepared in step (1);

[0049] (3) The ball mill was quick-frozen with liquid nitrogen, and then the 96-well deep-well plate in step (2) was placed in the module for grinding, and the parameters were set to P6, 30 Hz, 1 min, and repeated 2 times; the mixture was allowed to stand at room temperature for 5 min; and then centrifuged at 4000 rpm at room temperature for 3 min;

[0050] (4) Add 1-bromo-3-chloropropane to the 96-well plate in step (3), 200 μL per well;

[0051] (5) Place the 96-well deep-well plate from step (4) on a vortexer and vortex for 30 seconds; leave it at room temperature for 10 minutes; then centrifuge it at 4000 rpm, 4°C for 15 minutes;

[0052] (6) Take about 400 μL of the supernatant after centrifugation in step (5) to each well, then add 400 μL of isopropanol to each well, invert to mix, and place at -20°C for alcohol precipitation for no less than 30 min;

[0053] (7) Centrifuge the 96-well deep-well plate from step (6) at 4000 rpm, 4°C for 20 min;

[0054] (8) Discard the supernatant after centrifugation in step (7), and a white precipitate will be visible;

[0055] (9) Rinse the precipitate in step (8) once with 500 μL 75% ethanol and centrifuge at 4000 rpm, 4°C for 5 min;

[0056] (10) Discard the supernatant from step (9); place the 96-well deep-well plate in a centrifuge at 4000 rpm, 4°C, and centrifuge for 5 min;

[0057] (11) discarding the excess liquid after centrifugation in step (12), and drying the precipitate until it becomes translucent;

[0058] (12) Dissolve the precipitate with 50 μL RNase-free water, place the 96-well deep-well plate in a 60°C metal bath, and dissolve for 10 min;

[0059] (13) Total RNA concentration was measured by NanoDrop2000; 10 μL of total RNA was taken from each well into a 96-well PCR plate, and mRNA was enriched from the total RNA using vazyme mRNA capture beads;

[0060] (14) After mRNA enrichment, cDNA first-strand synthesis was performed. The reaction system in each well was as follows: 10 μL mRNA (10 ng-100 ng), 2 μL ABclonal oligo d(T) 23 VN Primer (50μM), 2μL 10×M-MμLV buffer, 1μL M-MuLV RT (200U / μL), 1μL 10mM dNTPs mix, 0.5μL RNase Inhibitor (40U / μL), 3.5μL RNase-free Water; place the above system on a PCR instrument and react according to the following program: 105℃, Lid; 42℃, 1h; 65℃, 20min;

[0061] (15) Prepare the following reaction system for Tn5 digestion (from vazyme TD501 kit): 4 μL TTEmix V50, 8 μL 5×TTBL, 20 μL of the reaction product in step (17), and 8 μL RNase-free water; place the above system on a PCR instrument and perform the reaction according to the following procedure: 105°C, Lid; 55°C, 10 min; 4°C, Hold.

[0062] (16) Add 80 μL of DNA Clean beads to each well of the 96-well plate in step (15), pipette to mix, and let stand at room temperature for 5 min;

[0063] (17) Discard the supernatant on the 96-well plate magnetic stand and add 200 μL 80% ethanol to each well to rinse the beads. Repeat twice.

[0064] (18) Discard excess ethanol and dry the beads; elute with 24 μL RNase-free water; collect the supernatant into a new 96-well PCR plate;

[0065] (19) Prepare the following system for PCR library amplification (from vazyme TD501 kit, amplification primers from vzayme TD203): 10 μL 5×TAB, 5 μL PPM, 1 μL TAE, 5 μL N6XX, 5 μL N8XX, 24 μL supernatant in step 21; place the above system on a PCR instrument and react according to the following program: 105°C, Lid; 72°C, 3 min; 98°C, 3 min; 98°C, 15 s, 60°C, 30 s, 72°C, 1 min, and the above temperatures for 14 cycles; 72°C, 5 min; 4°C, Hold;

[0066] (20) Add 0.55× DNAClean beads to the PCR product of step (19), pipette and mix; let stand at room temperature for 5 min;

[0067] (21) placing the 96-well PCR plate in step (20) on a magnetic stand, collecting the supernatant and placing it in a new 96-well PCR plate;

[0068] (22) Add 0.25× DNAClean beads to the supernatant collected in step (21), pipette and mix; let stand at room temperature for 5 min;

[0069] (23) Discard the supernatant on the 96-well PCR plate magnetic rack and add 200 μL 80% ethanol to each well to rinse the beads. Repeat twice.

[0070] (24) Discard excess ethanol and dry the beads; elute with 20 μL RNase-free water; collect the library supernatant into a new 96-well PCR plate;

[0071] (25) Qubit 3.0 was used to detect the concentration of mRNA-seq libraries, and the library yields were all above 100 ng ( Table 1 ), meeting the library input amount requirements for sequencing.

[0072] Table 1: Library yields.

[0073]

[0074] (26) The library was sent to Novogene for size detection and subsequent sequencing. The library fragment sizes were all between 200 and 600 bp ( Figure 8 ), which is similar to the library fragment size obtained by the Trueseq RNA-seq method. After sequencing on the Illumina platform, the sequencing data in Table 2 were obtained. The data in the table show that the library quality is high, but the read duplication ratio in the data is high, and the number of PCR amplification cycles can be appropriately reduced to 8-10.

[0075] Table 2: Sequencing data.

[0076] Library ID HRNA-seq.Rep1 HRNA-seq.Rep2 Material Fresh rice seedlings Fresh rice seedlings Library Type PE150 PE150 Raw Data Reads R1 97,161,516 65,122,811 Raw Data R1(Gbp) 14.5 9.7 Raw Data Reads 194,323,032 130,245,622 Raw Data(Gbp) 29.0 19.4 Clean Data Reads R1 92,821,734 60,064,414 Clean Data R1(Gbp) 9.4 5.2 Clean Data Reads 185,643,468 120,128,828 Clean Data(Gbp) 18.8 10.4 TrimmomaticPE Dropped 35.17％ 46.39％ Mapped Q30Reads 83,607,657 53,359,833 Q30Rate 45.04％ 44.42％ Unique Reads 92,779,077 62,412,961 Duplication Rate 43.03％ 39.78％

[0077] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library, characterized in that: The following steps are involved: (1) Grind the plant sample and Trizol together, let stand at room temperature, and then centrifuge at room temperature; (2) adding 1-bromo-3-chloropropane to the ground sample in step (1), vortexing, standing at room temperature, and then centrifuging at 4° C.; (3) taking the supernatant after centrifugation in step (2), adding isopropanol, inverting to mix, placing at -20°C for alcohol precipitation, and then centrifuging at 4°C; (4) Discard the supernatant after centrifugation in step (3), and a white precipitate will be visible; rinse the precipitate with 75% ethanol and then centrifuge at 4°C; (5) discarding the supernatant after centrifugation in step (4), and then centrifuging at 4°C; (6) discarding the excess liquid after centrifugation in step (5), drying the precipitate until it becomes translucent, and then dissolving the precipitate with RNase-free water; (7) Measure the total RNA concentration using NanoDrop2000; Take the total RNA into the PCR plate and use vazyme mRNAcapture beads to enrich mRNA from the total RNA; (8) cDNA single-strand synthesis; (9) performing Tn5 digestion; (10) Add DNA Clean beads, pipette to mix, and let stand at room temperature; (11) Discard the supernatant on the magnetic stand and add 80% ethanol to rinse the beads. Repeat twice. (12) Discard excess ethanol and dry the beads; elute with RNase-free water; collect the supernatant into a new PCR plate; (13) PCR library amplification; (14) Add 0.55× DNA Clean beads to the PCR product obtained in step (13), pipette and mix, and let stand at room temperature; then collect the supernatant on a magnetic rack and place it in a new PCR plate; (15) Add 0.25× DNAClean beads to the supernatant collected in step (14), pipette and mix, and let stand at room temperature; then discard the supernatant on the magnetic rack, add 80% ethanol to rinse the beads, and repeat twice; (16) Discard excess ethanol and dry the beads; elute with RNase-free water; collect the library supernatant into a new PCR plate; (17) Detect the concentration of mRNA-seq library.

2. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 1, characterized in that: In the step (1), a ball mill is used for grinding, the ball mill is quick-frozen with liquid nitrogen, a 96-well deep-well plate is used to place the material to be ground, and the material is allowed to stand at room temperature for 5 to 10 minutes; the centrifugal speed is 4000 to 5000 rpm, and the centrifugal time is 3 to 6 minutes.

3. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 2, characterized in that: In the step (2), the mixture is vortexed for 30 to 45 seconds and allowed to stand at room temperature for 10 to 18 minutes; the centrifugal speed is 4000 to 5000 rpm and the centrifugal time is 15 to 20 minutes.

4. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 3, characterized in that: The volume ratio of the supernatant after centrifugation and isopropanol in step (3) is 1:1, and the alcohol precipitation time is not less than 30 minutes; the centrifugal speed is 4000-5000 rpm, and the centrifugal time is 20-25 minutes; the centrifugal speed in step (4) is 4000-5000 rpm, and the centrifugal time is 5-10 minutes; the centrifugal speed in step (5) is 4000-5000 rpm, and the centrifugal time is 5-10 minutes.

5. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 4, characterized in that: In the step (6), the dissolution is carried out for 10 to 20 minutes, and the 96-well deep-well plate is placed in a metal bath at 60 to 70° C. for dissolution.

6. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 5, characterized in that: The reaction system in each well in step (8) is as follows: 10 μL mRNA (10 ng-100 ng), 2 μL oligo d(T) 23 VNPrimer (50μM), 2μL10×M-MuLV buffer, 1μL M-MuLV RT (200U / μL), 1μL 10mM dNTPs mix, 0.5μLRNase Inhibitor (40U / μL), 3.5μL RNase-free Water; place the reaction system on the PCR instrument and react according to the following program: Lid, 105℃; 42℃, 1h; 65℃, 20min.

7. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 6, characterized in that: In the step (9), the following reaction system is used for Tn5 digestion: 4 μL TTEmixV50, 8 μL 5×TTBL, 20 μL of the reaction product in step (9), and 8 μL RNase-free Water; the above system is placed on a PCR instrument and reacted according to the following program: 105° C., Lid; 55° C., 10 min; 4° C., Hold.

8. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 7, characterized in that: The step (13) uses the following reaction system to amplify the PCR library: 10 μL 5×TAB, 5 μL PPM, 1 μL TAE, 5 μL N6XX, 5 μL N8XX, 24 μL supernatant in step 21; the above system is placed on a PCR instrument and reacted according to the following program: 105°C, Lid; 72°C, 3 min; 98°C, 3 min; 98°C, 15 s, 60°C, 30 s, 72°C, 1 min, 8-11 cycles of the above temperatures; 72°C, 5 min; 4°C, Hold.

9. The method for preparing an efficient and high-throughput plant tissue mRNA-seq sequencing library according to claim 7, characterized in that: In step (10), the mixture is allowed to stand at room temperature for 5 to 10 minutes; and in step (14), the mixture is allowed to stand at room temperature for 5 to 10 minutes.

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