Double-chain connector for enriching full-length tRNA (transfer ribonucleic acid) and application of double-chain connector in tRNA sequencing

Abstract

The invention belongs to the technical field of high-throughput sequencing, and particularly relates to a double-chain connector for enriching full-length tRNA and application of the double-chain connector in tRNA sequencing. The double-chain joint comprises a 3'joint and a 5 'joint, and bases of the 3' joint and the 5 'joint are complementarily paired; the nucleotide sequence of the 3'linker is as shown in at least one of SEQ ID NO.1-4; and the nucleotide sequence of the 5 'linker is as shown in SEQ ID NO.5-8. According to the invention, the NGGU-sequence at the tail of the 5'joint in the double-chain joint is utilized, so that full-length tRNA can be enriched for sequencing; meanwhile, one random basic group is added to the 5'end of the linker, so that the preference of the linker to a certain tRNA molecule in the linker connection process is reduced. The double-chain connector is connected by a one-step method, so that the sample loss in experimental operation is reduced; and the double-chain connector is connected in 18h, so that the connection efficiency can be improved. Different samples are distinguished by using the double-chain header, so that mixed sample sequencing can be realized, the initial input amount is reduced, the sequencing cost is greatly reduced, the data yield is improved, and the expression dynamic state of tRNA can be better deeply analyzed.

Description

Technical Field

[0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a double-stranded adapter for enriching full-length tRNA and application thereof in tRNA sequencing. Background Art

[0002] tRNA plays an important role as a "bridge" connecting mRNA transcription and protein translation. tRNA has a complex secondary and tertiary structure. Its main function is to carry amino acids and participate in protein translation by pairing its anticodon with the codon on mRNA. There are many modification sites on tRNA, accounting for 78% of the 143 RNA modifications discovered. On average, about 11.9% of the nucleotides in each tRNA molecule are modified (DOI:10.1016 / j.tcb.2023.04.002), such as Figure 1 As shown in the literature, these modifications help maintain the structure of tRNA and regulate the abundance of tRNA in cells. In addition, tRNA modifications can change the swing of its base pairs, affect codon recognition, and affect ribosome occupancy, pauses, frameshift mutations in the translation process, and elongation steps to regulate translation speed and efficiency (DOI: 10.1016 / j.jgg.2022.10.002). The regulatory effects of modifications on tRNA on protein translation are as follows Figure 2 shown.

[0003] tRNA is encoded by multi-copy genes, and the sequences of tRNA molecules encoded by these genes are highly similar. Therefore, accurate full-length tRNA quantification methods are crucial to studying their expression dynamics. Early methods of quantifying tRNA by fluorescent labeling were difficult to distinguish highly similar tRNA molecules or detect modification information. With the development of high-throughput sequencing technology, second-generation sequencing platforms (such as Illumina) are able to generate a large amount of tRNA data, but during library construction, the reverse transcription process is susceptible to tRNA-rich modifications, resulting in mismatches or premature termination. Although the capture efficiency of full-length tRNA can be improved by removing methylation modifications and optimizing reverse transcriptase, the modification information cannot be retained, and the preference brought by PCR amplification affects the precise study of tRNA expression dynamics (DOI: 10.1016 / j.tig.2023.11.001).

[0004] Nanopore direct RNA sequencing developed by Oxford Nanopore is currently the only method that can directly sequence RNA. It can simultaneously obtain the sequence and modification information of RNA molecules without the need for PCR amplification. The principle is to drive the RNA single strand through the nanoscale protein pore with a constant voltage, resulting in current changes. Different bases or modification groups produce different current signals, thereby identifying the base sequence and modification site. At present, nanopore direct RNA sequencing is mostly used for the study of mRNA expression and modification. In 2021, the first study on the simultaneous application of nanopore direct RNA sequencing to tRNA quantification and modification was published, but it was only used to detect certain specific tRNA molecules, and the data yield was low (DOI: 10.1021 / acsnano.1c06488); in 2024, Nano-tRNAseq and data analysis methods for the dynamic study of yeast tRNA expression were developed, but their data yield is still low and the data analysis process is imperfect (DOI: 10.1038 / s41587-023-01743-6). At present, there is a lack of stable and reliable nanopore direct tRNA sequencing methods and perfect data analysis methods. The above two direct tRNA sequencing library construction methods are as follows Figure 3 shown.

[0005] Nanopore direct RNA sequencing can directly sequence RNA without PCR amplification, eliminating the influence of PCR bias and retaining the modification information on RNA. The library construction methods of the 2021 literature (DOI: 10.1021 / acsnano.1c06488) and "Nano-tRNAseq" both add 3' and 5' adapters to both ends of tRNA, open the secondary structure of tRNA through reverse transcription, and directly sequence tRNA molecules after connecting the nanopore sequencing dedicated adapter. Both technologies have certain defects and shortcomings:

[0006] (1) The 2021 paper (DOI: 10.1021 / acsnano.1c06488) only sequenced a few specific tRNA molecules in E. coli. The 3' and 5' adapters used were short, which easily led to unstable sequencing current signals of tRNA molecules in the early stages of sequencing. In addition, the final data output was low, the cost was high, and in-depth analysis was impossible.

[0007] (2) “Nano-tRNAseq” performed nanopore direct sequencing of tRNA in yeast. The direct RNA sequencing chip used was a low-version RNA002, which resulted in low data yield and the sequencing accuracy needed to be improved.

[0008] The library construction effects of both methods are not mature and stable enough, the sequencing chip version used is low, the data output is low, and the sequencing accuracy is not high enough. At present, Oxford Nanopore's direct RNA sequencing chip has been upgraded to RNA004, which has higher data output and greatly improved sequencing accuracy; the library construction process of both methods cannot perform mixed sequencing of multiple samples, and the sequencing cost is very high. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] The present invention discloses a double-stranded adapter for enriching full-length tRNA, wherein the double-stranded adapter comprises a 3' adapter and a 5' adapter, wherein the 3' adapter and the 5' adapter have complementary base pairing;

[0011] The nucleotide sequence of the 3' linker is at least one of SEQ ID NO.1-4:

[0012] GAUGGAAUCGUGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA (SEQ ID NO. 1);

[0013] GAUAUCAGCUAGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA (SEQ ID NO. 2);

[0014] GAUCUGGCAUAGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA (SEQ ID NO. 3);

[0015] GAUAGAUCUAGGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA (SEQ ID NO. 4);

[0016] The nucleotide sequence of the 5' linker is shown in SEQ ID NO.5-8:

[0017] CCUAAGAGCAAGAAGAAGCCACGAUUCCAUCUGGN(SEQ ID NO.5);

[0018] CCUAAGAGCAAGAAGAAGCCUAGCUGAUAUCUGGN (SEQ ID NO. 6);

[0019] CCUAAGAGCAAGAAGAAGCCUAUGCCAGAUCUGGN(SEQ ID NO.7);

[0020] CCUAAGAGCAAGAAGAAGCCCUAGAUCUAUCUGGN (SEQ ID NO. 8); N is one of the random bases A, T, G, C and U.

[0021] The invention discloses an application of a double-stranded adapter in tRNA sequencing.

[0022] The present invention also discloses a method for tRNA sequencing by enriching full-length tRNA through double-stranded adapters, the method comprising the following steps:

[0023] (1) Total RNA extraction, oxidation, purification and β-elimination;

[0024] (2) isolating tRNA based on step (1);

[0025] (3) removing 3' phosphorylation from the tRNA obtained in step (2);

[0026] (4) separating the tRNA after ligating the 3'-phosphate-depleted tRNA in step (3) to the double-stranded linker according to claim 1 or 2;

[0027] (5) Reverse transcription: Reverse transcription of the isolated tRNA obtained in step (4) is performed and the product is purified;

[0028] (6) After the sequencing adapters are connected, the product is purified and finally sequenced.

[0029] Preferably, in step (4), the connection method of the dual-link connector is:

[0030] (1) After the dissolved tRNA and double-stranded adapter are mixed, the following reagents are added in order:

[0031] 1.5ul 10x Annealing buffer

[0032] 1ul SuperaseIn

[0033] The total volume was 15ul, denatured at 90°C for 2min, and then decreased to 25°C at a rate of 0.1°C / sec;

[0034] (2) After the reaction, the product was immediately placed on ice and the following reagents were added in order:

[0035] 2ul 200mM MgCl2

[0036] 2.5ul RNALigase 2

[0037] 5ul RNALigase 2Reaction buffer

[0038] 15ul 50% PEG8000

[0039] 0.5ul Ultra Pure water 0.5ul

[0040] The total volume is 50ul, and the PCR instrument program is set as follows: 25℃2h→16℃16h→4℃insulation.

[0041] Preferably, in step (4), tRNA, 3' linker and 5' linker are mixed at a molar ratio of 1.2:1:1.

[0042] Preferably, the reverse transcription method in step (5) is:

[0043] (1) 8.5ul ultrapure water RNA, 3ul NEBNext Quick Ligation Reaction Buffer

[0044] 1ul RNase out, 1ul 1.4uM RT Adapter (RTA) and 1.5ul T4 DNA Ligase were mixed and incubated at 25℃ for 10min;

[0045] (2) Add 13ul Ultra Pure water, 3ul dNTPs (10mM each), 8ul 5x Induro RT reaction buffer and 2ul Induro Reverse Transcriptase to the system in step (1); the total volume is 40ul. Mix well and incubate at 60℃ for 30min, 70℃ for 10min, and keep warm at 4℃.

[0046] Beneficial effects of the present invention:

[0047] 1. The "NGGU-" sequence at the tail of the 5' linker in the double-stranded linker can be used to enrich the full-length tRNA for sequencing; at the same time, a random base is added to the 5' end of the linker to reduce the preference of the linker connection process for a certain tRNA molecule.

[0048] 2. Use one-step method to connect the double-chain connector to reduce sample loss during experimental operation; connect the double-chain connector for 18 hours to improve connection efficiency.

[0049] 3. Using the barcodes on the double-stranded adapter to distinguish different samples can achieve mixed sample sequencing, reduce the starting input, greatly reduce sequencing costs, increase data output, and better analyze the expression dynamics of tRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Modifications on tRNA;

[0051] Figure 2 Regulation of protein translation by modifications on tRNA;

[0052] Figure 3 Library construction flow chart of two nanopore direct tRNA sequencing methods;

[0053] Figure 4 Double-chain connector structure;

[0054] Figure 5 The database construction flow chart of this application;

[0055] Figure 6 Comparison of the yield of the present invention with "Nano-tRNAseq" and literature data;

[0056] Figure 7 Sublibrary tRNA read segment alignment diagram;

[0057] Figure 8 tRNA read length distribution of normal mouse liver tissue samples;

[0058] Fig. 9 Aminoacyl-tRNA coverage map of normal mouse liver tissue samples. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] The present invention uses two steps of oxidation purification and β elimination to remove the amino acid carried by tRNA or the A base of the CCA tail of the tRNA molecule that does not carry an amino acid during tRNA pretreatment, and reflects the situation of carrying amino acids through the CC / CCA ratio. In addition, the tRNA molecule is not subjected to demethylation treatment, and the modification on the tRNA is retained.

[0061] When performing adapter ligation, the double-stranded adapter used in the present invention is composed of a single-stranded 3' adapter and a 5' adapter. The double-stranded adapter structure of the present invention is as follows: Figure 4As shown. The 3' linker sequence is GAUGGAAUCGUGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA, and the 5' linker sequence is CCUAAGAGCAAGAAGAAGCCACGAUUCCAUCUGGN (the italic part is the Barcode; there are 4 sets of complementary single-stranded linkers, and the bases are the same except for the Barcode. The Barcodes of the other 3 sets of linkers are: AUCAGCUA, CUGGCAUA, and AGAUCUAG). The single-stranded 3' linker and the 5' linker form a double-stranded linker through complementary base pairing. The part connected to the 5' end of the tRNA molecule is designed as the "NGGU-" sequence. "GGU" can be complementary paired with the CCA tail of tRNA, which can specifically enrich the full-length tRNA for sequencing. At the same time, adding random bases can reduce the preference of the connection process; the polyadenylic acid part of the 3' linker sequence can be complementary paired with the primer of the subsequent reverse transcription step to ensure the smooth progress of the reverse transcription step. The double-stranded linker Barcode can be used to distinguish different samples, realize mixed sample sequencing, and reduce costs. The library construction effect and cost-effectiveness are much higher than the technologies involved in the above literature and "Nano-tRNAseq". Figure 5 As shown:

[0062] The experimental steps are as follows:

[0063] 1. RNA oxidation, purification and β-elimination

[0064] (I) Extraction of total RNA using Trizol

[0065] (II) RNA oxidation and purification

[0066] 1. RNA oxidation: according to the concentration, add an equal volume of freshly prepared NaIO4 (final concentration of 50mM) and react at 22℃ for 30min. Then add 1M glucose (final concentration of 100mM) and stop the reaction at room temperature for 5min;

[0067] 2. RNA purification: pass through Micro Bio Spin P30 columns, centrifuge at 1000g, room temperature for 4 minutes;

[0068] 3. The first round of anhydrous ethanol precipitation, stand at -80℃ for more than 3h;

[0069] 4. The second round of anhydrous ethanol precipitation was carried out at -80℃ for more than 3 hours.

[0070] (III) β elimination

[0071] 1. Dissolve RNA in 10ul ultrapure water for 7min;

[0072] 2. β-elimination of RNA

[0073] (1) Add freshly prepared sodium borate and react at 45°C for 90 min;

[0074] (2) Pass through a P30 column, centrifuge at 1000 g for 4 min at room temperature;

[0075] (3) Ethanol precipitation

[0076] 2. 15% TBU Gel to separate tRNA

[0077] 1. Dissolve tRNA in 10ul ultrapure water for 7min;

[0078] 2. Prepare 15% TBU polyacrylamide gel and pre-run at 180V for 15 minutes;

[0079] 3. Prepare samples: After the precipitate is dissolved, add an equal volume of 2× gel loading buffer, mix well, take 0.5ul Low range ssRNA marker, add 9.5ul UP water, then add 10ul 2× gel loading buffer, mix well;

[0080] 4. Place tRNA and marker in a 95℃ metal bath for 2-3 minutes, then immediately place on ice for 2-3 minutes; add sample, 200V, 90min.

[0081] 5. Gel staining: Use SYBR to stain the gel; cut out the tRNA bands of corresponding length (70-100nt) with a disposable blade and place them in nested tubes;

[0082] 6. Centrifuge the nested tubes at 13,000 rpm at room temperature for 5 minutes, add 400 ul RNA Elution Buffer and 1 ul SUPERase In to the 2.0 ml tube, and rotate on a rotator at room temperature for at least 7-8 hours (overnight) to allow tRNA to precipitate from the gel.

[0083] 7. Ethanol precipitation of tRNA: Precipitate with anhydrous ethanol and let stand at -80℃ for more than 3h.

[0084] 3. T4 PNK treatment - removal of 3' phosphate

[0085] 1. Dissolve tRNA in 10ul ultrapure water for 7min;

[0086] 2. Denaturation at 80℃;

[0087] 3. Prepare 24.5ul reaction system:

[0088] 2.5ul 10x T4PNK Reaction Buffer without ATP

[0089] 1ul SUPERase In

[0090] 1ul T4PNK

[0091] React at 37°C for 1 h, inactivate at 65°C for 10 min, and keep warm for 4 hours;

[0092] 4. Phenol extraction;

[0093] 5. Precipitate with anhydrous ethanol and let stand at -80℃ for more than 3h.

[0094] 4. Connect the dual link connector

[0095] The single-stranded 3' adapter sequence in the double-stranded adapter (the italic underlined part is the barcode):

[0096] (1) GAU GGAAUCGU GGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA (SEQ ID NO. 1);

[0097] (2) GAU AUCAGCUA GGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA(SEQ ID NO.2); (3)GAU C UGGCAUA GGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA(SEQ ID NO.3);

[0098] (4) GAU AGAUCUAG GGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAAA(SEQ ID NO.4);

[0099] Single-stranded 5'adapter sequence complementary to the double-stranded adapter (complementary by sequence number):

[0100] (1)CCUAAGAGCAAGAAGAAGCCACGAUUCCAUCUGGN (SEQ ID NO.5);

[0101] (2) CCUAAGAGCAAGAAGAAGCCUAGCUGAUAUCUGGN (SEQ ID NO. 6);

[0102] (3) CCUAAGAGCAAGAAGAAGCCUAUGCCAGAUCUGGN (SEQ ID NO. 7);

[0103] (4) CCUAAGAGCAAGAAGAAGCCCUAGAUCUAUCUGGN (SEQ ID NO. 8);

[0104] 1. Dissolve tRNA;

[0105] 2. After tRNA and single-stranded adapter are mixed at a molar ratio of 1.2:1:1, add the following reagents in order:

[0106] 10x Annealing buffer (1.5ul)

[0107] SuperaseIn(1ul)

[0108] 3. The total volume is 15ul, denature at 90℃ for 2min, and then decrease to 25℃ at a rate of 0.1℃ / sec;

[0109] 4. After the reaction, place the product on ice immediately and add the following reagents in order:

[0110] 200mM MgCl2 (2ul)

[0111] RNALigase 2 (dsRNA) (2.5ul)

[0112] RNALigase 2Reaction buffer(5ul)

[0113] PEG8000 (50%) (15ul)

[0114] Ultra Pure water (0.5ul)

[0115] 5. The total volume is 50ul. Set the PCR instrument program: 25℃2h→16℃16h→4℃insulation.

[0116] 5. 15% TBU Gel Separation of tRNA

[0117] 1. Dissolve tRNA in 10ul ultrapure water for 7min;

[0118] 2. Prepare 15% TBU polyacrylamide gel and pre-run at 180V for 15 minutes;

[0119] 3. Prepare samples: After the precipitate is dissolved, add an equal volume of 2× gel loading buffer, mix well, take 0.5ul Low range ssRNA marker, add 9.5ul UP water, then add 10ul 2× gel loading buffer, mix well;

[0120] 4. Place tRNA and marker in a 95℃ metal bath for 2-3 minutes, then immediately place on ice for 2-3 minutes; add sample, 200V, 90min.

[0121] 5. Gel staining: Use SYBR to stain the gel; cut out the tRNA (115-180nt) band with a disposable blade and put it into a nested tube;

[0122] 6. Centrifuge the nested tubes at 13,000 rpm at room temperature for 5 minutes, add 400 ul RNA Elution Buffer and 1 ul SUPERase In to the 2.0 ml tube, and rotate on a rotator at room temperature for at least 7-8 hours (overnight) to allow tRNA to precipitate from the gel.

[0123] 7. Ethanol precipitation of tRNA: Precipitate with anhydrous ethanol and let stand at -80℃ for more than 3h.

[0124] 6. Reverse Transcription

[0125] 1. Dissolve different tRNA samples in 8.5ul ultrapure water;

[0126] 2. Add the following reagents in sequence

[0127] 3ul NEBNext Quick Ligation Reaction Buffer

[0128] 1ul RNase out

[0129] 1ul RT Adapter(RTA)(1.4uM)

[0130] 1.5ul T4 DNA Ligase

[0131] After mixing, incubate at 25°C for 10 min.

[0132] 3. Add the following reagents in sequence

[0133] 13ul Ultra Pure water

[0134] 3ul dNTPs (10mM each)

[0135] 8ul 5x Induro RT reaction buffer

[0136] 2ul Induro Reverse Transcriptase

[0137] The total volume is 40ul. After mixing, incubate at 60℃ for 30min, 70℃ for 10min (PCR instrument), and keep warm at 4℃.

[0138] 7. Product Purification

[0139] 1. Add 72ul of resuspended Agencourt RNAClean XP beads (PCR tube) to the product after reverse transcription.

[0140] Incubate at 25°C for 5 min. Place the PCR tube on the magnetic stand, wait for 5 min, discard the supernatant, and wash the magnetic beads twice with 80% ethanol, with an interval of 30 s between each wash.

[0141] 2. After instant centrifugation to remove residual ethanol, add 23ul ultrapure water to resuspend the magnetic beads, put the PCR tube back on the magnetic rack and wait for 5 minutes, then take 23ul supernatant for the next reaction.

[0142] 8. Nanopore sequencing adapter ligation

[0143] Add the following reagents to the reverse transcription product in sequence:

[0144] 8ul NEBNext Quick Ligation Reaction Buffer

[0145] 6ul RNA Ligation Adapter(RLA)

[0146] 3ul T4 DNA Ligase

[0147] The total volume was 40ul, mixed and incubated at 25℃ for 30min.

[0148] IX. Product Purification

[0149] 1. Add 16ul of resuspended Agencourt RNAClean XP beads (PCR tube) to the product after the ligation reaction and incubate at 25℃ for 5min. Place the PCR tube on the magnetic rack, wait for 5 minutes, and discard the supernatant.

[0150] 2. Resuspend the magnetic beads with Wash Buffer (WSB) solution, place the PCR tube on the magnetic rack, wait for 5 minutes, and discard the supernatant. Repeat step 2 once.

[0151] 3. After instant centrifugation to remove the residual WSB solution, add 13ul RNA Elution Buffer (REB) to resuspend the magnetic beads, put the PCR tube back on the magnetic rack and wait for 5 minutes, take 12ul of the supernatant for the next reaction, and 1ul of the supernatant for quantification.

[0152] 10. Library sequencing

[0153] 1. Prepare the sequencing premix in advance and add the following reagents in order to 12ul of the purified mixed library:

[0154] 37.5ul Sequencing Buffer (SB)

[0155] 25.5ul Library Solution (LIS)

[0156] The total volume is 75ul. Mix well and place at room temperature for later use.

[0157] After adding 75ul of the mixed library to the chip, sequencing began. The sequencing chip version is RNA004.

[0158] Application Examples

[0159] Take the four tissue samples of normal mouse liver (Liver Normal), small intestine (SI Normal) and starved mouse liver (Liver Starvation), small intestine (SI Starvation) as examples. The data yield of the four sample mixed libraries is compared with "Nano-tRNAseq" and the literature (DOI: https: / / doi.org / 10.1101 / 2024.07.23.604651) Figure 6 As shown; the alignment of tRNA reads of each sub-library in the mixed library is shown Figure 7 As shown in Figure 2, taking normal mouse liver tissue as an example, the tRNA read length distribution and aminoacyl tRNA coverage are shown in Figure 2. Figure 8 , 9 As shown. Fig. 9 It can be seen that this method can better enrich the full-length tRNA to achieve mixed sequencing, greatly increase data output and reduce costs. In the future, the expression and modification of tRNA under different physiological conditions in different tissues can be analyzed, so as to more accurately and deeply study the dynamics of tRNA expression and its regulatory effect on protein translation.

Claims

1. A double-stranded adapter enriched with full-length tRNA, characterized in that: The double-stranded adapter comprises a 3' adapter and a 5' adapter, wherein the 3' adapter and the 5' adapter have complementary base pairing; The nucleotide sequence of the 3' linker is at least one of SEQ ID NO.1-4: (1) GAUGGAAUCGUGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA (SEQ ID NO. 1); (2)GAUAUCAGCUAGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA (SEQ ID NO. 2); (3)GAUCUGGCAUAGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA (SEQ ID NO. 3); (4) GUAAGAUCUAGGGCUUCUUCUUGCUCUUAGGAAAAAAAAAAAA (SEQ ID NO. 4); The nucleotide sequence of the 5' linker is shown in SEQ ID NO.5-8: (1) CCUAAGAGCAAGAAGAAGCCACGAUUCCAUCUGGN (SEQ ID NO. 5); (2) CCUAAGAGCAAGAAGAAGCCUAGCUGAUAUCUGGN (SEQ ID NO. 6); (3) CCUAAGAGCAAGAAGAAGCCUAUGCCAGAUCUGGN (SEQ ID NO. 7); (4) CCUAAGAGCAAGAAGAAGCCCUAGAUCUAUCUGGN (SEQ ID NO. 8); The N is one of the random bases A, T, G, C and U.

2. Use of the double-stranded adapter according to claim 1 in tRNA sequencing.

3. A method for tRNA sequencing by enriching full-length tRNA using the double-stranded adapter according to claim 1, characterized in that: The method comprises the following steps: (1) Total RNA extraction, oxidation, purification and β-elimination; (2) isolating tRNA based on step (1); (3) removing 3' phosphorylation from the tRNA obtained in step (2); (4) separating the tRNA after ligating the tRNA with 3' phosphate removed in step (3) to the double-stranded linker of claim 1; (5) Reverse transcription: Reverse transcription of the isolated tRNA obtained in step (4) is performed and the product is purified; (6) After the sequencing adapters are connected, the product is purified and finally sequenced.

4. The method according to claim 3, characterized in that In step (4), the connection method of the dual-link connector is: (1) After the dissolved tRNA and double-stranded adapter are mixed, the following reagents are added in order: 1.5ul 10x Annealing buffer 1ul SuperaseIn The total volume was 15ul, denatured at 90°C for 2min, and then decreased to 25°C at a rate of 0.1°C / sec; (2) After the reaction, the product was immediately placed on ice and the following reagents were added in order: 2ul 200mM MgCl2 2.5ul RNALigase 2 5ul RNALigase 2Reaction buffer 15ul 50% PEG8000 0.5ul Ultra Pure water 0.5ul The total volume is 50ul, and the PCR instrument program is set as follows: 25℃2h→16℃16h→4℃insulation.

5. The method according to claim 3, characterized in that: In step (4), tRNA, 3' linker and 5' linker are mixed at a molar ratio of 1.2:1:

1.

6. The method according to claim 3, characterized in that The method of reverse transcription in step (5) is: (1) 8.5ul ultrapure water RNA, 3ul NEBNext Quick Ligation Reaction Buffer 1ul RNase out, 1ul 1.4uM RT Adapter and 1.5ul T4 DNA Ligase were mixed and incubated at 25℃ for 10min; (2) Add 13ul Ultra Pure water, 3ul dNTPs, 8ul 5x Induro RTreaction buffer and 2ul Induro Reverse Transcriptase to the system in step (1); The total volume is 40ul. After mixing, incubate at 60℃ for 30min, 70℃ for 10min, and keep warm at 4℃.

7. The method according to claim 6, characterized in that The dNTPs included dATP, dGTP, dTTP and dCTP, each at a concentration of 10 mM.

Images