CN120843644A - A method for constructing a tRNA high-throughput sequencing library - Google Patents

Abstract

The present invention discloses a method for constructing a tRNA library, particularly a method for efficiently constructing a full-length tRNA high-throughput sequencing library. The method comprises the following steps: a) providing a sample containing small RNAs; b) deacylating the sample from step a); c) performing a 3′ dephosphorylation treatment on the deacylated product obtained from step b) to convert the 3′ phosphate and 3′ cyclic phosphorus of the RNA into 3′ hydroxyl groups; d) ligating the dephosphorylated product from step c) with an excess of a 3′ adapter to obtain a primary ligation product; e) reverse transcribing the primary ligation product using TGIRT as a reverse transcriptase to obtain a reverse transcription product containing cDNA; f) ligating the reverse transcription product with a 5′ adapter to obtain a secondary ligation product; and g) PCR amplifying the secondary ligation product to obtain a library containing full-length tRNAs. This method can produce a tRNA library with a high proportion of full-length tRNAs suitable for high-throughput sequencing. The present invention also relates to a method for high-throughput sequencing of full-length tRNAs, comprising constructing a tRNA library using the above method and then sequencing the constructed tRNA library.

Description

Method for constructing tRNA high-throughput sequencing library

Technical Field

The invention belongs to the fields of molecular biology and biotechnology, relates to a method for constructing a tRNA library, and in particular relates to a method for efficiently constructing a high-throughput sequencing library of full-length tRNA, wherein the ratio of the full-length tRNA to the tRNA is up to about 90%.

Background

Transfer RNAs (trnas) are a class of non-coding small RNA molecules that are widely present in organisms and are responsible for amino acid transfer during peptide chain synthesis, and are approximately 76-90 nucleotides (nt) in length. tRNA is about 4% -15% of the total RNA of the cell and plays a vital role in the protein translation and decoding process, and the main function is to translate codons on messenger RNA (mRNA) into protein. the metabolic function of tRNA is abnormal and can lead to the development of a variety of human diseases including muscle and nervous system diseases, cancer, immunodeficiency, lung and liver diseases, diabetes, etc. However, due to the lack of accurate, high-resolution methods for sequencing and quantification of tRNA, the regulation of tRNA levels and their physiological significance is still not fully understood. Thus, there is a need to establish efficient full length tRNA sequencing technology and use it to analyze cell and tissue specific tRNA composition in order to advance innovative research in traditional scientific questions, elucidating tRNA composition, tissue expression specificity and its function and mechanism in various diseases.

With the rapid development of high throughput sequencing technology, transcriptome sequencing has been widely used. Transcriptome sequencing generally refers to mRNA-Seq, i.e., the detection of the amount of mRNA expressed at the whole genome level that is capable of producing a protein. Full length tRNA molecules have very stable secondary structures and high levels of nucleotide modifications, which can severely impact the full length extension activity of reverse transcriptases during high throughput sequencing library construction and alignment of nucleic acid sequences (e.g., typical reverse transcriptases stall when encountering modifications on tRNA's, resulting in difficulty in obtaining full length tRNA sequences), and thus tRNA sequencing technology progress is very retarded.

The known methods for constructing libraries of tRNA's have various problems, such as quantitative mature tRNA sequencing (QuantM-tRNAseq) which do not overcome the reverse transcription barrier, the fact that the reverse transcription stops at the modification site, which results in the majority of the short sequences in the final library, and the hybridization-based method which can avoid the need for cDNA synthesis, but which can only distinguish tRNA's that differ by at least 8 nucleotides, which are not suitable for tRNA quantification, due to the very high sequence similarity between tRNA transcripts, which may differ by only one nucleotide even if different codons are read. Strategies to overcome structure and modification-induced reverse transcription termination include tRNA fragmentation, use of thermostable reverse transcriptases TGIRT (thermostable group II intron REVERSE TRANSCRIPTASE) with sustained synthesis capability and high fidelity, such as TGIRT-seq and DM-tRNAseq, and removal of part of the methylation modifications on tRNA with demethylase AlkB (ARM-seq and DM-tRNAseq). Although these methods have improved tRNA sequencing, there are still limitations in that the method using TGIRT usually requires the use of a combination of cyclase, which easily leads to intramolecular cyclization of the reverse transcription primer, results in a large number of adaptor self-ligating fragments in the library, and is expensive, while the problem with the use of the demethylase AlkB is the selectivity and damage of the AlkB treatment to tRNA. Moreover, all of these methods only alleviate a small portion of the reverse transcription arrest, which only restores tRNA types with specific methylation modifications, or those more sensitive to in vitro treatment with demethylase, while having no effect on other modified tRNA types.

Disclosure of Invention

Problems to be solved by the invention

The present invention is directed to a novel method for constructing a tRNA library that is capable of efficiently constructing a high proportion of full length tRNA's, which library is suitable for high throughput sequencing. Preferably, the method does not use the demethylase AlkB and/or cyclase, and thus solves, reduces or avoids tRNA damage, adaptor self-ligation and/or high cost issues associated therewith.

Means for solving the problems

In a first aspect, the invention provides a method of constructing a library of full length tRNAs, in particular a library of high throughput sequencing of full length tRNAs, comprising, consisting essentially of, or consisting of:

a) Providing a sample comprising a small RNA;

b) Deacylation of the sample of step a);

c) 3 'dephosphorization of the deacylated product obtained in step b) to convert 3' phosphate and 3 'cyclophosphates of the RNA into 3' hydroxyl groups;

d) Connecting the dephosphorization product obtained in the step c) with excessive 3' joints to obtain a primary connection product;

e) Performing reverse transcription on the primary ligation product by using TGIRT as reverse transcriptase to obtain a reverse transcription product containing cDNA;

f) Ligating the reverse transcription product with a 5' linker to obtain a secondary ligation product, and

G) PCR amplification of the secondary ligation product resulted in a library comprising full-length tRNA.

Wherein the sample comprising small RNAs provided in step a) can be obtained by extracting total RNAs from a biological sample and then isolating the small RNAs from the total RNA extract. Isolation of the small RNAs from the total RNA extract may be accomplished using purification isolation methods known in the art, provided that the desired small RNAs can be isolated from the total RNA. Preferably, the purification and separation described above can be performed using, for example, one or more of a Urea-PAGE gel, a glass fiber filter, a silica gel spin column, or a combination thereof. For example, the purification and isolation described above can be performed using, for example, the MEGA CLEAR kit available from Thermo (cat No. AM 1908). Preferably, the isolated small RNA is less than 100nt in length. Enrichment of small RNAs, particularly small RNAs less than 100nt, in this manner effectively decontaminates rRNA and enriches tRNA's, providing a relatively pure Input source (Input) for subsequent full-length tRNA library construction.

In step b), deacylation may be performed using Tris-HCl (Tris-hydroxymethyl aminomethane hydrochloride). For example, deacylation may be performed using about 100mM Tris-HCl (pH 9.0).

The 3' dephosphorization in step c) can be performed using a reagent capable of dephosphorizing the deacylated tRNA. For example, dephosphorization can be performed using T4 polynucleotide kinase (T4 PNK) without the addition of ATP, or using alkaline phosphatase.

The ligation reaction in step d) may be performed using RNA ligase known in the art. Preferably, the ligation reaction is performed using T4 RNA ligase 2, such as T4 RNA ligase 2 (truncated KQ) or T4 RNA ligase 2 (truncated K227Q) available from NEB company. In some embodiments, to increase ligation efficiency and reduce bias, an excess of 3' linker over the dephosphorization product, e.g., a 2-10 fold excess of 3' linker over the dephosphorization product, e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold excess of 3' linker is added. In some embodiments, after the ligation reaction of step d) is completed, the adenylation on the linker is removed first with a deacylase (e.g., a deacylase available from NEB under the designation M0331S) and then the excess 3' linker is cleared to reduce or avoid self-ligation of the linker. Preferably, 5'-3' exonuclease RecJf (e.g., 5'-3' exonuclease RecJf available from NEB under the accession number M0264L) is used to remove excess adaptors.

The reverse transcription in step e) may be performed using a thermostable group II intron reverse transcriptase, TGIRT enzyme (e.g. TGIRT ^TM -III from InGex). The reverse transcription is preferably carried out at a low temperature, wherein low temperature herein means a temperature of not higher than 60 ℃, e.g. in the range of 30-50 ℃, 35-45 ℃, 37-42 ℃, such as about 37 ℃, about 42 ℃, or about 50 ℃. Furthermore, the reverse transcription in step e) is preferably performed in a reaction buffer system with a low KCl salt concentration, in particular, the reverse transcription in step e) may be performed in a reaction system comprising 10-60mM Tris-HCl (pH about 7.5-8.5), 25-100mM KCl and 2-4mM MgCl ₂, more preferably, the reverse transcription may be performed in a reaction system comprising about 50mM Tris-HCl (pH about 8.3), 25-100mM KCl and about 3mM MgCl ₂. Even more preferably, the reverse transcription may be performed in a reaction system comprising about 50mM Tris-HCl (pH=8.3), about 75mM KCl and about 3mM MgCl ₂.

The reverse transcription of step e) may be carried out for a suitable time to obtain sufficient cDNA product. For example, reverse transcription can be performed for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so forth. Preferably, after reverse transcription is completed, excess reverse transcription primer is digested and then RNA is degraded to release reverse transcribed cDNA. Preferably, the excess reverse transcription primer is digested with 3'-5' single stranded DNA exonuclease Exo I. Preferably, the RNA is degraded by alkaline hydrolysis at elevated temperature.

The ligation reaction in step f) may be performed using RNA ligase known in the art. Preferably, the ligation reaction is performed using T4RNA ligase 1. More preferably, the ligation reaction is performed using a high concentration of T4RNA ligase 1 (e.g., available from NEB under the trade designation M0437M T RNA ligase 1;30 units/. Mu.L), and still more preferably overnight ligation is performed.

The PCR amplification in step g) can be performed using conditions and equipment deemed appropriate by the person skilled in the art, as long as a library comprising full-length tRNA can be obtained by this step.

In some embodiments, after one or more of steps b) -g) above, the resulting product is purified or ethanol precipitated. Specifically, in one embodiment, the product is purified after performing the deacylation of step b), preferably using an RNA Clean & Concentrator-5 kit (Zymo, R1016 or R1015) to replace the reaction buffer. This purification can also be replaced by ethanol precipitation, which takes longer than the RNA Clean & Concentrator-5 kit. In one embodiment, the product is purified after performing the 3' dephosphorization of step c), preferably using an RNA Clean & Concentrator-5 kit to replace the reaction buffer and remove proteins/enzymes. This purification can be replaced by ethanol precipitation, which takes longer than the RNA Clean & Concentrator-5 kit. In one embodiment, the primary ligation product is purified after performing the 3' ligation of step d), preferably using an RNA Clean & Concentrator-5 kit, to replace the reaction buffer and remove proteins/enzymes. This purification can be replaced by ethanol precipitation, which takes longer than the RNA Clean & Concentrator-5 kit. In one embodiment, the reverse transcription product is purified after performing the reverse transcription of step e), preferably using magnetic beads, more preferably MyONE Silane magnetic beads. In one embodiment, the secondary ligation product is purified after performing the 5' ligation of step f), preferably using magnetic beads, more preferably MyONE Silane magnetic beads. In one embodiment, the amplification products are purified and recovered after performing the PCR amplification of step g), preferably using magnetic bead purification, e.g.AMPure XP magnetic bead purification, and recovery of this step may be performed by agarose gel electrophoresis or polyacrylamide gel electrophoresis (PAGE), preferably by polyacrylamide gel electrophoresis to recover the amplification products, e.g.TBE-PAGE. Preferably, the amplified product is purified and recovered for isolation to yield a library comprising tRNA's of 180-220bp in length.

Preferably, the above-described methods of constructing the tRNA library do not use the demethylase AlkB and/or cyclase, or do not comprise a demethylation and/or cyclization step. More preferably, the above method of constructing a tRNA library does not use the demethylase AlkB and cyclase, or does not comprise a demethylation and cyclization step.

Preferably, the 5 'and 3' linkers used in the above methods of constructing the tRNA library comprise protecting groups to avoid inter-connection between linker molecules.

In one embodiment, the 3' linker used in the above method of constructing a tRNA library has the sequence 5' rAPP-GATCGGAAGAGCGTCGTG-3' SpC3 (SEQ ID NO: 1).

In one embodiment, the sequence of the 5' linker used in the above method of constructing a tRNA library is 5' phos-NNNNNNNNNNAGATCGGAAGAGCACACGTCTG-3' SpC3 (SEQ ID NO: 2).

In a second aspect, the invention provides a method of sequencing tRNA, wherein a library of tRNA's is constructed according to the method of the first aspect described above, and then the constructed library of tRNA's is sequenced. Sequencing may be performed using sequencing techniques or methods or platforms known in the art, e.g., may be performed usingThe system performs sequencing.

Effects of the invention

The method of the invention has one or more of the following advantages:

firstly, the substrate containing nucleic acid is not treated by AlkB, so that the selectivity and damage of the AlkB treatment to tRNA are avoided, and the integrity of the tRNA is ensured;

second, by using TGIRT such thermostable reverse transcriptase with sustained synthesis capability and high fidelity, reverse transcriptase is prevented from stopping at the modification site and crossing the modification site to obtain full length tRNA;

thirdly, adopting a linear library construction mode, namely respectively adding joints at two ends of RNA, avoiding using cyclase, effectively avoiding self-connection of the joints and having lower cost, simultaneously, designing protecting groups at the 5 'end and the 3' end of the joints, adding excessive first joints, removing excessive joints after the connection reaction is finished, digesting excessive/residual reverse transcription primers after reverse transcription, purifying by using magnetic beads after the connection of the second joints, and the like, thereby reducing the self-connection of the joints and improving the connection efficiency, and further ensuring the construction of a high-quality library for subsequent high-throughput sequencing;

Fourth, according to the methods of the invention, libraries containing high proportions (e.g., proportions greater than about 80%, greater than about 85%, greater than about 87%, or greater than about 90%, even up to 93%) of full-length tRNA can be obtained, and, even though the methods of the invention may have mismatches at the modification sites during reverse transcription, since the sites at which the mismatches occur are precisely the sites of modification, not only can full-length tRNA be obtained, but information about the modification sites on tRNA can be obtained from libraries of tRNA constructed using the invention, which is particularly important for most species for which no tRNA modification study has been conducted, and, in addition, most of the full-length tRNA can be obtained without bias.

Drawings

FIG. 1 is a flow chart illustrating a library construction method according to an embodiment of the present application;

FIG. 2 shows the proportion of each type of RNA in the library of tRNA's constructed in the examples of the application;

FIG. 3 shows the ratio of tRNAs or tRNAs fragments of various lengths in a library of tRNAs constructed in the examples of the present application;

FIG. 4 shows the ratio of tRNA lengths in a library of tRNA's constructed in the examples of the application.

Detailed Description

Definition of the definition

Unless otherwise indicated, terms used herein should be construed to have their ordinary meaning in the relevant art. Several terms used herein and their meanings are listed below.

As used herein, the term "sequencing" generally refers to methods and techniques for determining nucleotide base sequences in one or more polynucleotides. Polynucleotides may comprise nucleic acid molecules, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by a variety of systems in use, such as but not limited toPacific BiosciencesMGI、Complete Genomics、OxfordOr Life Technologies (Ion)) Is described. Alternatively or in addition, nucleic acid amplification, polymerase Chain Reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification may be used. Such systems can provide a plurality of raw genetic data corresponding to genetic information of a subject (e.g., animal, plant, microorganism, etc.), as generated by the system from a sample provided by the subject. In some examples, such systems provide sequencing reads (also referred to herein as "reads"). Reads may include a sequence of nucleobases corresponding to a nucleic acid molecule sequence that has been sequenced. Sequencing may include short read sequencing or long read sequencing, or both. In some cases, the systems and methods provided herein may be used with proteomic information.

As used herein, the term "small RNA" refers to an RNA molecule less than 200 nucleotides in length. The method mainly comprises non-coding RNA represented by micro RNA (miRNA), small interfering RNA (SMALL INTERFERING RNA, SIRNA), transfer RNA (TRANSFERRNA, TRNA) and the like.

As used herein, the term "high throughput sequencing" generally refers to a technique of sequencing a plurality of, e.g., up to hundreds of thousands to millions of nucleic acid molecules (DNA or RNA) at a time, which may also be referred to as next generation sequencing technology (next generation sequencing, NGS). High throughput sequencing allows sequencing to be done on a larger scale than Sanger sequencing (or first generation sequencing).

As used herein, the term "library" refers to a collection of nucleic acid fragments.

The term "sample" as used herein generally refers to a biological sample from a subject that includes a substance of interest (e.g., tRNA), where the subject can be an animal, such as a mammal (e.g., a human) or a bird (e.g., bird), or other organism, a plant (e.g., rice, maize, soybean), or a microorganism (e.g., bacteria, fungi, archaea, viruses). The biological sample may comprise a number of macromolecules, such as cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample may include one or more cells. The sample may include one or more microorganisms. The biological sample may be a nucleic acid sample or a protein sample. The sample may be a cell-free sample. The cell-free sample may comprise extracellular polynucleotides. The sample may be enriched prior to processing.

As used herein, the term "linker" and derivatives thereof generally refer to any linear oligonucleotide that can be attached to a nucleic acid molecule of the present disclosure. In some embodiments, the linker is not substantially complementary to the 3 'or 5' end of any target sequence present in the sample. In some embodiments, suitable linker lengths are in the range of about 10-60 nucleotides, about 15-50 nucleotides, about 15-40 nucleotides, about 15-35 nucleotides, about 20-30 nucleotides in length.

The term "about" as used herein is understood to be within the normal tolerance of the art, e.g., within 2 standard deviations of the mean, unless specified or apparent from the context. "about" is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numbers provided herein are modified by the term "about".

The ranges provided herein are to be understood as shorthand for all numbers within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subranges selected from 1、2、3、4、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、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、 or 50, and all fractional values between the above integers (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9). Regarding sub-ranges, specifically considered are "nested sub-ranges" that extend from any end point within the range. For example, nested subranges of the exemplary ranges of 1-50 can include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in another direction.

The invention is further illustrated below in conjunction with specific examples. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Examples

The routes of acquisition for the various materials and reagents described in the examples below are merely provided for the purpose of fully disclosing the source of acquisition for the materials and reagents of the invention and should not be construed as limiting. In fact, the sources and access of the materials and reagents used are broad, and materials and reagents that are not accessible against law and ethics (including biological materials) may be used instead in accordance with the concepts of the present invention. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. Furthermore, the nucleotide or amino acid sequences referred to in the examples may be synthesized by known techniques.

The sequencing platform employed in this example was an illuminea sequencing platform. For this sequencing platform, the inventors designed and synthesized the following linker and primer sequences:

In the sequences shown in the above table, rAPP is adenylated, phos is a phosphorylation modification, spC is 3'spacer C3, N is any one of the 4 nucleotides A, T, G and C, which are random nucleotides inserted during primer synthesis, and the 10 consecutive random nucleotides are included in the 5' linker to remove PCR repeats during library amplification.

In Index primers 1 to 3 of the above table, the portion shown underlined is the six base tag sequence "IIIIII", where I represents one nucleotide of the 4 nucleotides A, T, G and C. The tag sequences contained in the Index primers are used to label (i.e., PCR amplification with different Index primers for different samples or libraries) different samples or libraries in a multi-sample mixed library of a sequencing platform, so that the different samples or libraries can be distinguished later, i.e., the tag sequences contained in each individual Index primer are defined six base sequences, but the tag sequences contained in different Index primers are different from each other to distinguish, and the other sequences contained in the Index primers are identical to each other except for the tag sequences. The present example uses Index primers 1-3 shown in the above table for 3 different samples, but it will be understood by those skilled in the art that other Index primers differing only in the tag sequence can be constructed and synthesized according to the actual situation and requirements.

FIG. 1 is a flow chart illustrating a library construction method according to an embodiment of the present application. Hereinafter, for purposes of illustration and not limitation, a process is described for constructing a full length tRNA high throughput sequencing library from a rice spike sample and sequencing by a sequencing platform according to the methods of the application to identify protein-bound RNA species and RNA sites in plants.

1. Extraction of Total RNA

1) In this example, 3 different rice spike samples were taken, 100mg each was ground to a fine powder in 3 different mortar, 1mL TRIzol (Thermo, 15596018) was added, and mixed well each;

2) The mixture was centrifuged at 12000g for 10 min at 4℃and the pellet was discarded;

3) Transferring the supernatant to a new centrifuge tube, adding 200 mu L of chloroform, oscillating for 15 seconds, and standing for 5 minutes;

4) Centrifuging the solution after standing at 4 ℃ for 10 minutes with 12000g, and carefully sucking the upper aqueous phase;

5) Adding 1.5 times of isopropanol, uniformly mixing, and standing at-20 ℃ for 30 minutes;

6) Centrifuging at 4 ℃ for 30 minutes at 12000g, and discarding the supernatant;

7) The pellet was washed with 70% ethanol, centrifuged at 4 ℃ for 30 minutes at 12000g, all supernatant carefully removed and dried at room temperature for 5 minutes;

8) Adding 300 mu L DEPC-H ₂ O to resuspend and deposit, and uniformly mixing by gun suction;

9) Adding an equal volume of phenol imitation (150 mu L of water saturated phenol and 150 mu L of chloroform), shaking and mixing for 15 seconds, and standing for 3 minutes;

10 4 ℃ and 12000g for 10 minutes, carefully sucking the upper water phase, adding 30 mu L of 3M sodium acetate (pH 5.2) and 3 times of absolute ethyl alcohol, and mixing well;

11 Standing for more than 30 minutes at a temperature of between 20 ℃ and 20 ℃;

12 4 ℃,12000g for 30 minutes, and discarding the supernatant;

13 1mL of 70% ethanol, centrifugation at 12000 Xg for 10 min at 4℃and careful removal of all supernatants and drying at room temperature for 5 min;

14 30-50. Mu.L DEPC-H ₂ O is added for re-suspension precipitation, RNA concentration is measured under the condition of A260 by using a Nanodrop or other spectrophotometry after sucking and mixing uniformly, when A260/A280 approaches 2, the purity of the RNA is higher, and the total RNA extract obtained by the method is used for the next step.

2. Isolation and purification of small RNAs

Small RNAs of less than 100nt were isolated and purified from the total RNA extract obtained in the previous step using MEGA CLEAR kit (Thermo, AM 1908). The maximum loading of the cartridge column was 500 μg.

1) Adding 5-500 μg total RNA (50 μg total RNA in this example) into elution buffer to 100 μl, and mixing by blowing;

2) Adding 350 mu L of binding buffer solution, blowing and uniformly mixing;

3) Adding 250 mu L of absolute ethyl alcohol, blowing and uniformly mixing;

4) The mixture was transferred to a filter tube and centrifuged at 12000g for 30 seconds;

5) Retaining flow through (RNA is smaller than 100nt in flow through, so that most rRNA and mRNA can be effectively removed), adding 3 mu L glycogen (Thermo, R0551), 45 mu L3M sodium acetate (pH 5.2), 900 mu L absolute ethyl alcohol, and standing at-20 ℃ for more than 1 hour;

6) Centrifuging at 13000rpm for more than 30 min at 4 ℃, and discarding the supernatant;

7) The pellet was washed with 1mL of 75% ethanol, centrifuged at 13000rpm for 10 minutes at 4℃and the supernatant discarded;

8) Air-drying at room temperature for 5 minutes;

9) Resuspension in 50-100 μl of DEPC water, measuring RNA concentration with Nanodrop or other spectrophotometry under a260 conditions;

10 200ng of small RNA is taken for subsequent tRNA library construction, and the rest of small RNA is stored in a refrigerator with the temperature of-80 ℃ for standby.

3. Deacylation of purified small RNAs

Since tRNA will be added with amino acid by aminoacyl tRNA synthetase, tRNA is deacylated by treatment with Tris-HCl prior to library construction. RNA obtained in the previous step and Tris-HCl (pH 9.0) were added according to the following table, mixed well and left to react for 45 minutes at 37 ℃.

	Volume (mu L)
		RNA(200ng)+H2O	18
1M Tris-HCl pH9.0	2

4. Recovery of deacylated RNA

The deacylated product obtained in the previous step was recovered using an RNA Clean & Concentrator-5 kit (Zymo, R1016).

All centrifugation is carried out at 10,000-16,000 g.

Pretreatment of RNA washing buffer 48ml of absolute ethanol was added to 12ml of washing buffer (Wash buffer) contained in the kit when the kit was first used.

1) Adding water to the 3 RNA samples to make up to 50 mu L respectively, adding 2 times of volume (100 mu L) of RNA binding buffer solution, and uniformly mixing;

2) Adding 600 mu L of absolute ethyl alcohol, and uniformly mixing;

3) Transferring into a collection pipe, centrifuging for 30 seconds, and discarding the flow through;

4) Adding 400 mu L RNA Prep Buffer, centrifuging for 30 seconds, and removing the flow through;

5) Adding 700 mu L of the pretreated RNA washing buffer, centrifuging for 30 seconds, and removing the flow-through;

6) 400. Mu.L of the pretreated RNA wash buffer was added and centrifuged for 2 min, and the column was transferred to a new dorf tube;

7) The RNA was dissolved by adding 11. Mu.L of DEPC-treated water to the center of the column, left at room temperature for 2min, centrifuged for 30 seconds, and the column was discarded.

Dephosphorization treatment of T4-PNK

The recovered product from the previous step was dephosphorized using T4-PNK (NEB, M0201L).

1) Adding the following reagents into 10 mu L of RNA sample, and uniformly mixing;

Reagent(s)	Volume (mu L)
		10 XPNK buffer	2
T4-PNK	1
		RiboLock RNase Inhibitor(Thermo,EO0381)	1
H2O	6

2) The mixed system was incubated at 37 ℃ for 1 hour;

3) PNK heat inactivation was performed by incubation at 65 ℃ for 20 minutes.

6. Recovery of dephosphorized RNA

The product from the previous step was subjected to RNA recovery using the RNA Clean & Concentrator-5 kit (Zymo, R1016).

1) Adding water to each sample to make up to 50 μl, adding RNA binding buffer times volume (100 μl), mixing

2) Adding 600 mu L of absolute ethyl alcohol, and uniformly mixing;

3) Transferring into a collection pipe, centrifuging for 30 seconds, and discarding the flow through;

4) Adding 400 mu L RNA Prep Buffer, centrifuging for 30 seconds, and removing the flow through;

5) Adding 700 mu L RNA Wash Buffer, centrifuging for 30 seconds, and removing the flow through;

6) Add 400 μ L RNA Wash Buffer, centrifuge for 2min, transfer column to new dorf tube;

7) The RNA was dissolved by adding 7. Mu.L of DEPC-treated water to the center of the column, left at room temperature for 2min, centrifuged for 30 seconds, and the column was discarded.

7.3' Joint connection

1) Adding 2 mu L of 20 mu M3' joint into the RNA sample obtained in the previous step, and uniformly mixing;

2) Denaturation at 82 ℃ for 2min, immediately placing on ice;

3) The 3' linker ligation mixtures were prepared according to the following table, where the reagents required were from T4 RNA LIGASE 2, truncated KQ (NEB, M0373L);

4) Add 12. Mu.L of the above mixture to each RNA sample, blow well with a gun, ligate for 3 hours at 25 ℃;

5) Adaptor de-adenylation, namely adding 1 mu L of 5' de-adenylate enzyme (NEB, M0331S) into the connection product, blowing and mixing uniformly, and reacting for 1h at 30 ℃;

6) Degrading the unconnected 3' joint, namely adding 2 mu L of RecJF exonuclease (NEB, M0264L) into the reactant, blowing and mixing uniformly, and reacting for 1h at 37 ℃;

7) Heat inactivation-incubation at 70℃for 20 min.

8. Recovery of RNA with 3' linker attached

The ligation product obtained in the previous step was recovered using an RNA Clean & Concentrator-5 kit.

1) Adding water to the 3 RNA samples to make up to 50 mu L respectively, adding 2 times of volume (100 mu L) of RNA binding buffer solution, and uniformly mixing;

2) Adding 600 mu L of absolute ethyl alcohol, and uniformly mixing;

3) Transferring into a collection pipe, centrifuging for 30 seconds, and discarding the flow through;

4) Adding 400 mu L RNA Prep Buffer, centrifuging for 30 seconds, and removing the flow through;

5) Adding 700 mu LRNA of washing buffer, centrifuging for 30 seconds, and removing flow-through;

6) 400. Mu.L of RNA wash buffer was added and centrifuged for 2 min, and the column was transferred to a new dorf tube;

7) The RNA was dissolved by adding 10. Mu.L of DEPC-treated water to the center of the column, left at room temperature for 2min, centrifuged for 30 seconds, and the column was discarded.

TGIRT-III reverse transcription

1) Adding 2 mu L of 4 mu M reverse transcription primer (RT primer) into 9 mu LRNA samples, and blowing and mixing uniformly;

2) Denaturation at 82 ℃ for 2min, immediately placing on ice;

3) Reverse transcription mixtures were prepared according to the following table:

5 XLow salt buffer 250mM Tris-HCl pH8.3, 37mM KCl,15mM MgCl ₂;

4) Adding the mixture in the step 3) into the RNA sample and the reverse transcription primer in the step 2), and uniformly mixing. Reverse transcription at about 50℃for 16 hours;

5) Reverse transcriptase inactivation, incubation at 80 ℃ for 10min;

6) Removing the excess reverse transcription primer by adding 1 mu L Exonuclease I (NEB, M0293S) to the reverse transcription product and digesting at 37℃for 30 min;

7) 1 mu L of 0.5M EDTA is added into the enzyme digestion reaction to terminate the reaction;

8) Adding 5 mu L of 1M NaOH, uniformly mixing, taking out a gun head, remaining on pH test paper, detecting, wherein the pH is about 11, 95 ℃ for 3min, and immediately placing on ice;

9) After neutralization by adding 4.5. Mu.L of 1M HCl, the mixture was mixed and the tip was left on a pH test paper to detect the mixture, and the pH was about 8.

Purification of cDNA by MyONE Silane magnetic beads

MyONE Silane was used commercially available from Thermo under the trade designation 37002D.

1) 3 Samples are respectively corresponding to a dorf mL low-adsorption tube, 10 mu L MyONE Silane magnetic beads are added into a new low-adsorption dorf tube, separated on a magnetic rack, and the supernatant is removed;

2) Washing the beads with 500. Mu.L of RLT buffer (QIAGEN, 79216), separating on a magnetic rack, and discarding the supernatant;

3) The beads were resuspended with 94.5. Mu.L of RLT buffer;

4) Mixing magnetic beads uniformly, adding the mixture into a reverse transcription product to be purified, and sucking and beating the mixture uniformly;

5) Adding 114 mu L of absolute ethyl alcohol, sucking, beating and uniformly mixing;

6) Standing at room temperature for 5min, and sucking and beating for 2 times;

7) Separating on a magnetic frame, and discarding the supernatant;

8) Washing the magnetic beads with 1mL of 80% ethanol, transferring the magnetic beads to a new tube, separating on a magnetic rack, and removing the supernatant;

9) Washing the beads with 1mL of 80% ethanol again, separating on a magnetic rack, and removing the supernatant;

10 Centrifugation at 800rpm for 3 seconds, removal of residual droplets, air drying on a magnetic rack for 5 minutes;

11 The beads were resuspended with 5. Mu.L of 5mM Tris-HCl (pH 7.5).

11.5' Joint connection

1) Adding 1.3 mu L of 100 mu M5' joint and 1 mu L of DMSO into the magnetic beads, sucking and beating, and uniformly mixing;

2) Denaturation at 82 ℃ for 2min, immediately placing on ice;

3) The 5' linker ligation mixtures were prepared according to the following table, in which reagents were all from RNALIGASE 1,High concentration (NEB, M0437M)

Mixing, adding 12.7 μl of the above mixture into each sample, and mixing with gun;

4) The block was heated at 25 degrees, 1300rpm, for 1 minute every 10 minutes, for 16 hours.

Purification of cDNA by MyONE Silane magnetic beads

1) 3 Samples are respectively corresponding to a dorf mL low-adsorption tube, 5 mu L MyONE Silane magnetic beads are taken, separated on a magnetic rack, and the supernatant is removed;

2) Washing the magnetic beads with 500 μl of RLT buffer, separating on a magnetic rack, and discarding the supernatant;

3) The beads were resuspended with 60. Mu.L of RLT buffer;

4) Mixing magnetic beads uniformly, adding the mixture into a connection product connected with a 5' connector, and sucking and beating the mixture uniformly;

5) Adding 60 mu L of absolute ethyl alcohol, sucking, beating and uniformly mixing;

6) Standing at room temperature for 5min, and sucking and beating for 2 times;

7) Separating on a magnetic frame, and discarding the supernatant;

8) Washing the beads with 1mL 80% ethanol by transferring the beads to a new tube, separating on a magnetic rack, and removing the supernatant;

9) Washing the beads with 1mL of 80% ethanol again, separating on a magnetic rack, and removing the supernatant;

10 Centrifugation at 800rpm for 3 seconds, removal of residual droplets, air drying on a magnetic rack for 5 minutes;

11 The beads were resuspended in 55. Mu.L of 10mM Tris-HCl (pH 7.5), separated on a magnetic rack at 1300rpm on a 25℃heating block for 5min, and 50. Mu.L was aspirated into a new tube.

PCR amplified library

1) PCR mixtures were prepared according to the following table, with 3 libraries corresponding to one of Index primers 1-3, respectively.

Reagent(s)	Volume (mu L)
		Q5,High-Fidelity 2×PCR Master Mix(NEB,M0492S)	25
Universal primers	1
		Index primer 1/2/3	1
cDNA	15
		H2O	8

2) Setting up a PCR instrument according to the procedure of the following table

14. Magnetic bead purification of PCR amplified library

The amplification product obtained in step 13 was purified using AMPure XP magnetic beads (available from Beckman, A63881). This step can also be performed using Northey DNA Clean loads (N411-02).

1) Adding 2x (namely 90 mu L) AMPure XP magnetic beads into the amplified PCR products, uniformly mixing, and standing for 5 minutes at room temperature;

2) Separating on a magnetic rack for 5 minutes;

3) Carefully aspirate the supernatant and leave a bit of liquid to avoid aspiration of the beads;

4) Washing the beads with 200 μl of 80% ethanol, and removing the supernatant;

5) Repeating the washing once, carefully removing all residual liquid;

6) Drying for 5 minutes at room temperature on a magnetic frame;

7) Taking the PCR tube off the magnetic frame, adding 15 mu L of water to resuspend the magnetic beads, and standing for 5 minutes at room temperature;

8) The magnetic rack was separated, 14. Mu.L of supernatant was pipetted into a new PCR tube and 3. Mu.L of 6 XDNA loading buffer was added.

TBE-PAGE gel library collection

1) PCR products were purified using 6% TBE-PAGE gel

A6% TBE-PAGE gel was prepared according to the following table, 5mL of 0.75mm thick mini-slab was required for 1 block, two 10mL blocks, and the formulation shown in the following table was 10mL, and 2 gels were suitable for formulation.

2) 135V, electrophoresis for 50 min, staining with SYBR ^TM Safe DNA gel dye (Thermo, S33102), and recovering the gel according to the following steps;

3) Cutting the strip of about 180-220bp with a blade into a prepared 600 mu L low adsorption EP tube (the bottom of which is provided with 1-2 small holes by a 10mL syringe burnt by an alcohol lamp), sleeving 600 mu L of EP tube into a 2mL low adsorption EP tube, centrifuging at 12000rpm for 5 minutes, and grinding the rubber strip;

4) After centrifugation, 600. Mu.L of the EP tube was discarded, and 300μl DNA gel extraction buffer(DNA gel extraction buffer:300mM NaCl,10mM Tris-HCl pH 8.0,1mM EDTA),55℃ sol was added to 2mL of the EP tube containing the PAGE gel fragments for 1-2 hours;

5) Transferring the crushed gum into a centrifugal filtration column Filtration Column (CORNING, CLS 8163), centrifuging at 12000rpm for 2min to collect liquid, and discarding the centrifugal column;

6) 2. Mu.L glycogen (Thermo, R0561), 30. Mu.L 3M sodium acetate (pH 5.2) and 1mL absolute ethanol were added sequentially, mixed well and precipitated at-80℃for 1 hour to overnight;

7) Taking out the precipitated product at-80 ℃, and centrifuging at 12000rpm in a refrigerated centrifuge precooled to 4 ℃ for 30min;

8) Carefully remove the supernatant, note that no white precipitate was aspirated. 1ml of freshly prepared 70% ethanol was added to the EP tube and centrifuged at 12000rpm for 10min in a refrigerated centrifuge pre-cooled to 4 ℃;

9) Carefully remove the supernatant, note that no white precipitate was aspirated. Collecting residual liquid on the pipe wall to the bottom of the pipe by short centrifugation, carefully removing the residual liquid, uncovering and drying at room temperature for about 5 minutes;

10 15. Mu.L of Nuclear-free H ₂ O was added to dissolve the precipitate.

16. High throughput sequencing

Sequencing was performed in PE150 mode using the Nova-seq platform, 5G data volume per library. This time there were three samples. The high throughput analysis showed that tRNA accounted for more than about 90% of all reads (FIG. 2) and full length tRNA accounted for 87% -93% of all tRNA reads (FIGS. 3 and 4) for two biological replicates per sample.

The FINE-tRNA-seq library obtained according to the procedure described above in this example has the sequence shown below (wherein A, B, C, the tag sequence and the linker "-" between D are added for ease of reading only and do not represent any particular structure or linkage between these sequences in the actual library):

A-B-C-six base tag sequence-D,

Wherein A is AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO: 8), B is the sequence of the tRNA negative strand, C is NNNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (SEQ ID NO: 9), the six base tag sequence is IIIIII (as defined above), and D is ATCTCGTATGCCGTCTTCTGCTTG (SEQ ID NO: 10).

Methods for identifying RNA species and RNA sites that bind to proteins in plants have been described above by way of example. Although the embodiments have been described with reference to specific exemplary embodiments, it will be evident that various modifications and changes may be made to these examples without departing from the broader scope of the inventive subject matter. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Claims (10)

1. A method of constructing a library of full length trnas, the method comprising the steps of:

a) Providing a sample comprising a small RNA;

b) Deacylation of the sample of step a);

c) 3 'dephosphorization of the deacylated product obtained in step b) to convert 3' phosphate and 3 'cyclophosphates of the RNA into 3' hydroxyl groups;

d) Connecting the dephosphorization product obtained in the step c) with excessive 3' joints to obtain a primary connection product;

e) Performing reverse transcription on the primary ligation product by using TGIRT as reverse transcriptase to obtain a reverse transcription product containing cDNA;

f) Ligating the reverse transcription product with a 5' linker to obtain a secondary ligation product, and

G) PCR amplification of the secondary ligation product resulted in a library comprising full-length tRNA.

2. The process according to claim 1, wherein the process does not use or comprises a demethylase AlkB and/or a cyclase, preferably the process does not use a demethylase AlkB and a cyclase and does not comprise a demethylase and a cyclase.

3. The method of claim 1, wherein the 3 'linker used in step d) and the 5' linker used in step f) comprise protecting groups to avoid inter-connection between linker molecules.

4. A process according to any one of claims 1-3, wherein the obtained product is purified after one or more or all of steps b) -g), e.g. after all of steps b) -g).

5. A method according to any of claims 1-3, wherein an excess of 3' linkers, e.g. a 2-to 10-fold excess of 3' linkers, e.g. at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold excess of 3' linkers, relative to the dephosphorized product is added in step d).

6. A method according to any one of claims 1 to 3, wherein after the ligation reaction of step d) is completed, the adenylation on the 3 'linker is removed with a deacylase and the excess 3' linker is cleared with a 5'-3' single stranded DNA exonuclease.

7. A process according to any one of claims 1 to 3, wherein the reverse transcription in step e) is carried out at a low temperature of not more than 60 ℃.

8. The method of any one of claims 1-3, wherein the amplification product is purified and recovered after step g) to isolate trnas having a length of 180-220 bp.

9. A method of high throughput sequencing of full length tRNA, wherein the method comprises constructing a library of tRNA's according to the method of claim 1, and then sequencing the constructed library of tRNA's.

10. The method of claim 9, wherein use is made ofThe system performs sequencing.