CN113512767B

CN113512767B - Joint and kit for constructing small RNA library and construction method of small RNA library

Info

Publication number: CN113512767B
Application number: CN202110486484.5A
Authority: CN
Inventors: 李新; 马玉; 李瑞强; 赵桂仿
Original assignee: Tianjin Novogene Biological Information Technology Co ltd
Current assignee: Tianjin Novogene Biological Information Technology Co ltd
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-09-23
Anticipated expiration: 2041-04-30
Also published as: CN113512767A

Abstract

The invention discloses a joint and a kit for constructing a small RNA library and a construction method of the small RNA library. Wherein the linker comprises a 3 'end linker, the 3' end linker is SEQ ID NO: 1, wherein N represents a random base; the 5 'end of the 3' end joint is connected with an rApp, the 3 'end is connected with a blocking group, and the second base of the 5' end is modified with MP. Further, the linker also comprises a 5 'end linker, wherein the 5' end linker is SEQ ID NO: 2, the 3' end of the 5' end linker has a 2 ' OMe modification. By applying the technical scheme of the invention, MP modification of the 3' end connector and 2 ' OMe modification of the 5' end connector can effectively prevent the 3' end connector and the 5' end connector from being connected, improve the connector connection efficiency and reduce the initial quantity of database building.

Description

Joint and kit for constructing small RNA library and construction method of small RNA library

Technical Field

The invention relates to the technical field of biology, in particular to a joint and a kit for constructing a small RNA library and a construction method of the small RNA library.

Background

Small RNA mainly refers to Small RNA with the length of 20-30nt, including miRNA, snRNA, snorRNA, piwiRNA and the like, and plays a very important role in gene regulation. In recent years, with the development of high-throughput sequencing technology, the types, expression levels and functional characteristics of small RNA are continuously explored. Currently, standard small RNA pooling can be referred to NEB pooling kits (NEB # E7300S) starting with 1 μ g total RNA, by first performing 3 'end linker ligation followed by hybridization blocking with reverse transcription primers to the 3' end linker to prevent the residual 3 'end linker from generating non-specific dimers in the 5' end linker ligation reaction; and then, connecting 5' -end connectors, synthesizing cDNA through reverse transcription, performing polyacrylamide gel electrophoresis (PAGE) after PCR amplification, cutting gel of a library region, recovering and purifying to obtain the final on-machine library.

The prior art has the defects that the initial database building amount is high, the database building amount is generally 1 mu g, and the database building method is not suitable for building a database of rare species samples; reverse transcription primer blocking is not ideal, so that 5' -end connector connection reaction generates nonspecific connection; more nonspecific amplification is generated in the PCR amplification process, so that the yield of the library is low; before the machine is operated, the rubber needs to be cut, recovered and purified, and the time for rubber preparation, rubber running and rubber recovery is long, so that the time for building the whole warehouse is prolonged.

Disclosure of Invention

The invention aims to provide a joint for constructing a small RNA library, a kit and a method for constructing the small RNA library, and aims to solve the technical problem that the initial amount of small RNA library construction is too high in the prior art.

To achieve the above object, according to one aspect of the present invention, there is provided an adaptor for constructing a small RNA library. The linker comprises a 3 'end linker, wherein the 3' end linker is SEQ ID NO: 1, wherein N represents a random base; the 5 'end of the 3' end joint is connected with an rApp, the 3 'end is connected with a blocking group, and the second base of the 5' end is modified with MP.

Further, the linker also comprises a 5 'end linker, wherein the 5' end linker is SEQ ID NO: 2, the 3' end of the 5' end linker has a 2 ' OMe modification.

According to another aspect of the invention, a kit for constructing a small RNA library is provided. The kit comprises the above linker.

Further, the kit also comprises a 3' end connector connection system reagent and a 5' end connector connection system reagent, wherein the 3' end connector connection system reagent comprises an RNAse inhibitor, a truncated T4 ligase2, 10-30% PEG8000 and 10-30% DMSO.

Further, the 5' end linker ligation system reagent comprises 1 XT 4 ligase1 buffer, RNAse inhibitor, T4 ligase1 and ATP, wherein the 1 XT 4 ligase1 buffer comprises 50mM Tris-HCl, 10mM MgCl ₂ And 1mM DTT, pH 7.5.

Further, the kit also comprises a nucleic acid sequence as shown in SEQ ID NO: 3.

Further, the kit also comprises a nucleic acid sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and PCR amplification primers shown in the specification.

According to a further aspect of the invention, a method for constructing a small RNA library is provided. The construction method comprises the following steps: and (3) acquiring a small RNA sample, and constructing the small RNA library by adopting any one of the kits for constructing the small RNA library.

Further, the construction of the small RNA library by using any one of the kits for constructing the small RNA library comprises the following steps: 3' end joint connection: preparing a 3' end connector connecting mixed solution by adopting a 3' end connector connecting system reagent to connect small RNA and a 3' end connector to obtain the small RNA connected with the 3' end connector, wherein the 3' end connector connecting mixed solution comprises 10U-40U of RNAse inhibitor, 100U-300U of truncated T4 ligase2 or truncated T4 ligase 2KQ, 10-30% of PEG8000, 10-30% of DMSO and 1 x Buffer; 5' end joint connection: preparing a 5' end connector connection mixed solution by adopting a 5' end connector connection system reagent to connect a small RNA connected with a 3' end connector and the 5' end connector to obtain a small RNA connected with the 3' end connector and the 5' end connector, wherein the 5' end connector connection mixed solution comprises 1 XT 4 ligase1 buffer, 10-40U RNAse inhibitor, 10U-30U T4 ligase1 and 1 mM-20 mM ATP; reverse transcription reaction: using SEQ ID NO: 3, carrying out reverse transcription on small RNA connected with a 3 'end joint and a 5' end joint by using a reverse transcription primer pair to obtain cDNA; purification of the cDNA product: purifying cDNA by using magnetic beads, and removing dimers; PCR amplification and purification: library amplification was performed using Q5 Mix with amplification primers as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, and a PCR amplification primer; magnetic beads were used for purification.

Further, the 3' end linker is incubated at 20-37 ℃ for 10 minutes to 2 hours, preferably 1 hour, and enzyme inactivation is carried out at 65-85 ℃, preferably 65 ℃; preferably, the connection condition of the 5' end connector is incubation for 10 min-2 h at 25-28 ℃, enzyme inactivation for 20min at 65-85 ℃, and standing for 5min at 4 ℃; preferably, the reaction conditions for PCR amplification are: the first stage is as follows: at 95 ℃ for 3 min; and a second stage: 15 cycles of 98 ℃ for 10s, 65 ℃ for 30s and 72 ℃ for 30 s; and a third stage: 72 ℃ for 2 min.

By applying the technical scheme of the invention, MP modification of the 3' end connector and 2 ' OMe modification of the 5' end connector can effectively prevent the 3' end connector and the 5' end connector from being connected, improve the connector connection efficiency and reduce the initial quantity of database building.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a diagram illustrating a library building process according to an embodiment of the present invention;

FIG. 2 shows the detected peak pattern of the bovine-9 library in example 1;

FIG. 3 shows the detected peak pattern of the bovine-10 library in example 1;

FIG. 4 shows the detected peak pattern of the bovine-11 library in example 1;

FIG. 5 shows the detected peak pattern of the bovine-12 library in example 1;

FIG. 6 shows a graph of the detected peaks of the bovine-13 library in example 1;

FIG. 7 shows a graph of the detection peaks of the bovine-14 library in example 1;

FIG. 8 shows the detected peak pattern of the bovine-15 library in example 1;

FIG. 9 shows the detected peak pattern of the bovine-16 library in example 1;

FIG. 10 shows the detection peak pattern of the human-1 library in example 2;

FIG. 11 shows a graph of the detection peaks of the human-2 library in example 2;

FIG. 12 shows the detection peak pattern of the human-3 library in example 2;

FIG. 13 shows a graph of the detection peaks of the human-4 library in example 2;

FIG. 14 shows the detection peak pattern of the mouse-1 library in example 2;

FIG. 15 shows graphs of detection peaks of the mouse-2 library in example 2;

FIG. 16 shows the detection peak pattern of the mouse-3 library in example 2; and

FIG. 17 shows the detection peak pattern of the mouse-4 library in example 2.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

Aiming at the technical problem of overhigh initial amount of small RNA library construction in the prior art, the invention provides the following technical scheme.

According to an exemplary embodiment of the invention, a linker for constructing a small RNA library is provided. The linker comprises a 3 'end linker, wherein the 3' end linker is SEQ ID NO: 1, wherein N represents a random base; the 3' end linker is connected with an rApp at the 5' end and a blocking group (the blocking group can be ddC, NH) at the 3' end ₂ Spacer C12, Spacer C6, Spacer C3, etc.), and MP is modified at the second base at the 5' -end.

Wherein rApp is adenylation (adenylation); MP is methylphosphonate; 2 ' OMe is 2 ' -O-methyl, 2 ' methoxy. MP modification of the 3' end connector and 2 ' OMe modification of the 5' end connector can effectively prevent the 3' end connector and the 5' end connector from being connected, improve the connector connection efficiency and reduce the initial quantity of the library building.

For convenience of operation, according to an exemplary embodiment of the invention, a kit for constructing a small RNA library is provided. The kit comprises the above linker. Preferably, the kit further comprises a 3' end linker ligation system reagent and a 5' end linker ligation system reagent, the 3' end linker ligation system reagent comprising an RNAse inhibitor, truncated T4 ligase2, 10% to 30% PEG8000, 10% to 30% DMSO and 1 x Buffer. Among them, truncated T4 ligase2 and 1 XBuffer can be reagents in NEB pooling kit (NEB # E7300S).

In an exemplary embodiment of the invention, the 5' end linker ligation system reagent comprises 1 XT 4 ligase1 buffer, RNAse inhibitor, T4 ligase1 and ATP. Wherein, 1 XT 4 ligase1 buffer and T4 ligase1 can be the reagents in NEB banking kit (NEB # E7300S).

Preferably, the kit further comprises a nucleic acid sequence as set forth in SEQ ID NO: 3, the reverse transcription primer has high reverse transcription efficiency and good specificity. Further, the kit also comprises a nucleic acid sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and PCR amplification primers shown in the specification. The pair of amplification primers has high amplification efficiency and good specificity.

According to a further aspect of the invention, a method for constructing a small RNA library is provided. The construction method comprises the following steps: and (3) acquiring a small RNA sample, and constructing the small RNA library by adopting any one of the kits for constructing the small RNA library. Because MP modification of the 3' end connector and 2 ' OMe modification of the 5' end connector can effectively prevent the 3' end connector and the 5' end connector from being connected, thereby improving the connector connection efficiency and reducing the initial quantity of library construction.

Preferably, the construction of the small RNA library by using any one of the above-mentioned kits for constructing the small RNA library comprises the following steps: 3' end joint connection: preparing a 3' end connector connecting mixed solution by using a 3' end connector connecting system reagent to connect small RNA and a 3' end connector to obtain the small RNA connected with the 3' end connector, wherein the 3' end connector connecting mixed solution comprises 10U-40U RNAse inhibitor, 100U-300U truncated T4 ligase2 or truncated T4 ligase 2KQ (T4 ligase 2truncated KQ of NEB), 10-30% of PEG8000, 10-30% of DMSO and 1 xBuffer (for example, the small RNA can be obtained by diluting from NEB and T4 ligase 2truncated kit); 5' end joint connection: preparing a 5' end connector connection mixed solution by adopting a 5' end connector connection system reagent to connect a small RNA connected with a 3' end connector and the 5' end connector to obtain a small RNA connected with the 3' end connector and the 5' end connector, wherein the 5' end connector connection mixed solution comprises 1 XT 4 ligase1 buffer, 10-40U RNAse inhibitor, 10U-30U T4 ligase1 and 1 mM-20 mM ATP; reverse transcription reaction: using SEQ ID NO: 3, carrying out reverse transcription on small RNA connected with a 3 'end joint and a 5' end joint by using a reverse transcription primer pair to obtain cDNA; purification of the cDNA product: purifying cDNA by using magnetic beads, and removing dimers; PCR amplification and purification: library amplification was performed using Q5 Mix with amplification primers as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, PCR amplification primers shown in the specification; magnetic beads were used for purification.

The joint connection system is matched with the connection of the 3 'end joint and the 5' end joint, so that a good technical effect can be achieved. Thereby further improving the joint connection efficiency and reducing the initial amount of library construction. In addition, according to the invention, PAGE gel recovery is not needed, magnetic beads are used for purification, the library construction time is shortened while the library yield is improved, and the invention provides a small RNA library construction method with no gelatinization and low initial quantity (for example, starting with 100ng of total RNA).

In order to further improve the efficiency of the linker ligation, it is preferred that the conditions for the 3' end linker ligation are 20-37 ℃ for 1 hour incubation and 65 ℃ enzyme inactivation; preferably, the connection condition of the 5' end connector is incubation for 10 min-2 h at 25-28 ℃, enzyme inactivation for 20min at 65-85 ℃, and standing for 5min at 4 ℃. In a preferred embodiment of the present invention, the reaction conditions for PCR amplification are: the first stage is as follows: at 95 ℃ for 3 min; and a second stage: 15 cycles of 98 ℃ for 10s, 65 ℃ for 30s and 72 ℃ for 30 s; and a third stage: 72 ℃ for 2 min.

In a preferred embodiment of the invention, the 3' end fitting connects: performing linker ligation using a modified linker containing a random base, the reaction solution comprising: 20U RNAse inhibitor, 200U T4 ligase 2truncated, 20% PEG8000, 20% DMSO and 1 XBuffer; reaction conditions are as follows: incubating at 28 ℃ for 1 hour, and inactivating enzyme at 65 ℃; 5' end joint connection: the ligation is performed using a modified linker containing a random base, and the reaction solution includes: 1 XT 4 ligase1 buffer, 20U RNAse inhibitor, 20U T4 ligase1, 10mM ATP etc.; reaction conditions are as follows: incubating at 28 deg.C for 10min, inactivating enzyme at 65 deg.C for 20min, and standing at 4 deg.C for 5 min; reverse transcription to synthesize cDNA: cDNA was synthesized by reverse transcription using HiScript III Enzyme Mix under the following conditions: incubating at 25 deg.C for 5min, incubating at 50 deg.C for 45min, and inactivating enzyme at 85 deg.C for 5 s; purification of the cDNA product: purification using 0.4 × and 1.4 × magnetic beads to remove dimers;

PCR amplification and purification: library amplification was performed using Q5 Mix, reaction conditions: the first stage is as follows: at 95 ℃ for 3 min; and a second stage: 10s at 98 ℃, 30s at 65 ℃ and 30s at 72 ℃ for 15 cycles; and a third stage: 2min at 72 ℃; purification was performed using a combination of 1.3 × and 1.8 × magnetic beads.

The following examples are provided to further illustrate the advantageous effects of the present invention. Reagents or methods not specifically indicated in the present invention may be carried out using reagents or methods conventional in the art.

Example 1

See fig. 1 for a library building process.

In this example, the linker and primer information are shown in Table 1.

TABLE 1

The basic operation steps are as follows:

1.3' end linker and the reaction mixture is shown in Table 2 below.

TABLE 2

Total RNA samples were taken, diluted to 1. mu.l (100ng) and incubated at 70 ℃ for 2 min.

Mu.l of the sample was added to the mixture shown in Table 2 in total of 10. mu.l, and the reaction conditions are shown in Table 3.

TABLE 3

Temperature of	Time
		28℃	1h
65℃	20min
		4℃	At least 5min

The reaction was immediately followed to the next step.

2.5' end linker ligation reactions

The 5' end linker ligation reaction was prepared as in Table 4 below.

TABLE 4

Note: the 5' end of the joint was incubated at 70 ℃ for 2min before ligation.

The reaction conditions are shown in Table 5.

TABLE 5

Temperature of	Time
		28℃	10min
65℃	20min
		4℃	At least 5min

Immediately after the reaction, the reverse transcription was performed.

3. Reverse transcription was performed using the Novozan HiScriptIII 1ststrand synthesis cDNA kit.

The reverse transcription reagent was removed, dissolved at room temperature and the system as shown in Table 6 below was prepared on ice:

TABLE 6

Heating at 65 deg.C for 5min, rapidly cooling on ice, and standing on ice for 2 min.

4. First strand cDNA synthesis reaction solution was prepared, as shown in Table 7:

TABLE 7

Reagent	Volume of
		The mixed liquid of the previous step	20μl
10×RT Mix	2.5μl
		HiScript III Enzyme Mix	2.5μl
In total	25μl

Gently beat and mix with a pipette.

The first strand cDNA synthesis reaction was performed under the following conditions, see Table 8:

TABLE 8

25℃	5min
		50℃	45min
85℃	5sec

5. Magnetic bead chip selection (the next holy bead cargo number: 12600ES08)

(1) The RT product was added with water to 50. mu.l, and 20. mu.l of next sacred magnetic beads (0.4X) were added thereto, followed by mixing, and then the mixture was left at room temperature for 5 minutes. The supernatant was left to stand on the magnetic stand for 5 minutes until it became completely clear.

(2) The entire clarified supernatant was transferred to a new tube. Add 50. mu.l beads (final 1.4X) to the supernatant and mix well and let stand at room temperature for 5 minutes. The mixture was allowed to stand on a magnetic stand for 5 minutes, and the supernatant was discarded.

(2) After washing with 80% ethanol and air-drying, the column was eluted with 24. mu.l of nuclease-free water.

PCR amplification reaction

The Q5 mix (NEB, M0492L) system and procedure are shown in Table 9.

TABLE 9

Reagent	Volume of
		cDNA sample (No nuclease Water elution product in step 5)	23μl
Q5 mix buffer	25μL
		F primer (SEQ ID NO: 4) (10. mu.M)	1μL
R primer (SEQ ID NO: 5) (10. mu.M)	1μL
		In total	50μl

The reagents in Table 9 above were mixed well and reacted under the conditions shown in Table 10.

Watch 10

PCR product purification of 100-and 200-bp fragment (1.3 × → 1.8 ×) using Whole gold magnetic beads (EC411-03)

(1) To 50. mu.l of the PCR product, 65. mu.l of magnetic beads (1.3X) were added, gently shaken 10 times, and mixed, followed by standing at room temperature for 5 min. Standing on a magnetic frame for 5 min.

(2) The entire clarified supernatant was transferred to a new tube.

(3) Add 25. mu.l beads to the supernatant (final 1.8X), gently blow 10 times and mix well, and let stand at room temperature for 5 min. Standing on a magnetic frame for 5 min.

(4) Washing and elution were carried out with 20. mu.l of nuclease-free water.

This example was conducted in a detailed investigation of experimental conditions, such as: the concentration of the 3' end linker and the ligation reaction conditions, whether the 3' end linker is closed, the concentration of the 5' end linker and the ligation reaction time, the removal and purification of the dimer after reverse transcription, and the like are specifically shown in the following table 11:

TABLE 11

TABLE 12

FIG. 2 shows a detected peak pattern of a bovine-9 library, FIG. 3 shows a detected peak pattern of a bovine-10 library, FIG. 4 shows a detected peak pattern of a bovine-11 library, FIG. 5 shows a detected peak pattern of a bovine-12 library, FIG. 6 shows a detected peak pattern of a bovine-13 library, FIG. 7 shows a detected peak pattern of a bovine-14 library, FIG. 8 shows a detected peak pattern of a bovine-15 library, and FIG. 9 shows a detected peak pattern of a bovine-16 library.

The samples of the main peaks of the two independent libraries of cow-15 and cow-16 are subjected to on-machine sequencing, the analysis results are shown in table 12, the data such as Q20, Q30 and mapping rate are normal, the detection number of mature miRNA and precursor miRNA is normal, and the method can realize low-initial-amount and non-gelatinization library building.

Example 2

Comparing the effect of PEG8000 and DMSO addition on the experimental results, this example lists the results of the pooling of human and mouse samples after addition of both reagents, as shown in Table 13 below:

watch 13

Sample(s)	Initial amount (ng)	PEG8000 concentration	DMSO concentration	Results of library examination
					Person 1	500	-	-	Miscellaneous peaks
Person 2	500	20％	-	Miscellaneous peaks
					Person
3	500	-	20％	Miscellaneous peaks
					Person 4	500	20％	20％	Library peaks
Mouse 1	500	-	-	Library peaks
					Mouse 2	500	20％	-	Library peaks
Mouse
	3	500	-	20％	Library peaks
Mouse 4						500	20％	20％	Library peaks

Fig. 10 shows a human-1 library detection peak pattern, fig. 11 shows a human-2 library detection peak pattern, fig. 12 shows a human-3 library detection peak pattern, fig. 13 shows a human-4 library detection peak pattern, fig. 14 shows a mouse-1 library detection peak pattern, fig. 15 shows a mouse-2 library detection peak pattern, fig. 16 shows a mouse-3 library detection peak pattern, and fig. 17 shows a mouse-4 library detection peak pattern.

The experimental results show that the library results are significantly different due to the addition of PEG8000 and DMSO in the library construction process of different species, the human sample library construction has a large influence on the library results under the conditions of no addition, PEG8000 addition, DMSO addition and simultaneous addition of two reagents (the results are shown in figures 10-13), the library results of a mouse sample are small (the results are shown in figures 14-17), and the simultaneous addition of PEG8000 and DMSO also has a certain positive effect on the removal of linker dimer of the library as seen by comparing library inspection graphs of mice 1-3 and mouse 4; as can be seen from the peak diagrams of the library detection of human-4 and mouse-4 samples, the simultaneous presence of PEG8000 and DMSO is an important condition for the stable library establishment of different species, and the data results are shown in the following Table 14. Therefore, the simultaneous addition of PEG8000 and DMSO can effectively reduce the formation of a linker dimer in the library construction process, and is more beneficial to the stable library construction.

TABLE 14

Example 3

For soybean, human, murine and rice samples, the NEB kit and the method described in example 1 were used for pooling respectively, and the two methods were compared for pooling results as shown in table 15 below:

watch 15

The initial amounts of the NEB kit and the method in the embodiment 1 are respectively 1000ng and 100ng, the database construction quality of the two methods is good, but the detection quantity of the method in the embodiment 1 is relatively small in the detection of mature miRNA and precursor miRNA, the main reason is that the initial amount of the database construction is low, and the detection of partial miRNA with extremely low expression is difficult; different species, same sequencing depth, coverage difference. The data show that the method is highly consistent with the NEB kit in terms of data quality and data result, is between the characteristics of low initial amount and non-colloid library establishment of the method, and is more suitable for high-throughput industrialized production.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:

1) the modified joint and the optimized connection reaction condition can improve the specific connection efficiency, so that the initial amount of library construction is reduced to 100 ng;

2) removing cDNA dimers of non-specific connecting products after reverse transcription by using a magnetic bead purification method, and improving the content of a specific template in PCR amplification so as to improve the yield of a library;

3) the whole library building process is simple to operate, the processes of glue making, glue running and glue cutting recovery are not needed, the library building can be completed within 8 hours, the time is shortened by 0.5-1 day compared with a gel cutting recovery library building method, the library building flux is improved, the library building time is shortened, and the method is more suitable for the library building in an industrialization mode.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. An adaptor for use in the construction of a small RNA library, wherein said adaptor comprises a 3 'end adaptor, said 3' end adaptor being SEQ ID NO: 1, wherein N represents a random base; the 5 'end of the 3' end joint is connected with an rApp, the 3 'end is connected with a blocking group, and the second base of the 5' end is modified with MP; the linker further comprises a 5 'end linker, wherein the 5' end linker is SEQ ID NO: 2, the 5' end linker having a 2 ' OMe modification at the 3' end.

2. A kit for constructing a small RNA library comprising the linker of claim 1.

3. The kit of claim 2, further comprising a 3' end linker ligation system reagent and a 5' end linker ligation system reagent, said 3' end linker ligation system reagent comprising an RNAse inhibitor, truncated T4 ligase2, 10% to 30% PEG8000, and 10% to 30% DMSO.

4. The kit of claim 3, wherein the 5' end linker ligation system reagents comprise 1 XT 4 ligase1 buffer, RNAse inhibitor, T4 ligase1 and ATP, wherein the 1 XT 4 ligase1 buffer comprises 50mM Tris-HCl, 10mM MgCl ₂ And 1mM DTT, pH 7.5.

5. The kit of claim 2, further comprising the nucleotide sequence set forth in SEQ ID NO: 3.

6. The kit of claim 5, further comprising a nucleic acid sequence as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, and PCR amplification primers shown in the specification.

7. A method for constructing a small RNA library, comprising: obtaining a small RNA sample, and constructing a small RNA library by using the kit for constructing the small RNA library according to any one of claims 2 to 6.

8. The method for constructing the small RNA library according to claim 7, wherein the method for constructing the small RNA library by using the kit for constructing the small RNA library as described in any one of claims 2 to 6 comprises the following steps:

3' end joint connection: preparing a 3' end connector connecting mixed solution by using a 3' end connector connecting system reagent to connect a small RNA and a 3' end connector to obtain a small RNA connected with the 3' end connector, wherein the 3' end connector connecting mixed solution comprises 10U-40U of an RNAse inhibitor, 100U-300U of truncated T4 ligase2 or truncated T4 ligase 2KQ, 10-30% of PEG8000 and 10-30% of DMSO;

5' end joint connection: preparing a 5' end connector connection mixed solution by using a 5' end connector connection system reagent to connect the small RNA connected with the 3' end connector and the 5' end connector to obtain the small RNA connected with the 3' end connector and the 5' end connector, wherein the 5' end connector connection mixed solution comprises 1 XT 4 ligase1 buffer, 10-40U RNAse inhibitor, 10U-30U T4 ligase1 and 1 mM-20 mM ATP;

reverse transcription reaction: using SEQ ID NO: 3, carrying out reverse transcription on the small RNA connected with the 3 'end joint and the 5' end joint by using a reverse transcription primer to obtain cDNA;

purification of the cDNA product: purifying the cDNA by using magnetic beads, and removing dimers;

PCR amplification and purification: library amplification was performed using Q5 Mix with amplification primers as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, PCR amplification primers shown in the specification; magnetic beads were used for purification.

9. The method for constructing the recombinant vector DNA of claim 8, wherein the 3' end linker is connected under the conditions of incubation at 20-37 ℃ for 10 minutes to 2 hours and enzyme inactivation at 65-85 ℃.

10. The construction method according to claim 8, wherein the 5' end joint is connected under the conditions of incubation at 25-28 ℃ for 10-2 hours, enzyme inactivation at 65-85 ℃ for 20min, and standing at 4 ℃ for 5 min.

11. The method for constructing a nucleic acid molecule according to claim 8, wherein the reaction conditions for PCR amplification are: the first stage is as follows: at 95 ℃ for 3 min; and a second stage: 15 cycles of 98 ℃ for 10s, 65 ℃ for 30s and 72 ℃ for 30 s; and a third stage: 72 ℃ for 2 min.