阅读说明：本技术 一种基于随机序列利用载体筛选活性增强子的方法 (Method for screening active enhancer by using vector based on random sequence ) 是由 张玉波 朱秀生 黄雷 李清 于 2019-06-19 设计创作，主要内容包括：本发明公开了一种增强子筛选表达载体及利用随机序列筛选增强子的方法所述增强子筛选表达载体由穿梭质粒载体改造而成,增强子筛选表达载上的核心部件包括mini启动子及其下游的mcherry报告基因,mini启动子上游包含随机序列的重组位点。本发明提供一种增强子筛选表达载体及利用随机序列筛选增强子的方法,该方法利用增强子筛选表达载体和筛选文库实现对增强子序列的有效鉴别,相较于现有技术,该方法对基因组中增强子的鉴定准确率大幅提升,同时该方法能够针对几十bp序列中的增强子进行有效鉴定,对于深入理解基因表达调控的机制具有重要意义。(The invention discloses an enhancer screening expression vector and a method for screening an enhancer by using a random sequence, wherein the enhancer screening expression vector is formed by transforming a shuttle plasmid vector, the core component on the enhancer screening expression vector comprises a mini promoter and a mcherry reporter gene at the downstream of the mini promoter, and the upstream of the mini promoter comprises a recombination site of the random sequence. The invention provides an enhancer screening expression vector and a method for screening an enhancer by using a random sequence, wherein the method utilizes the enhancer to screen the expression vector and screen a library to realize effective identification of an enhancer sequence.)

1. The enhancer screening expression vector is characterized in that the enhancer screening expression vector is used for screening an active enhancer in a random sequence, the enhancer screening expression vector is formed by transforming a shuttle plasmid vector, core components on the enhancer screening expression vector comprise a mini promoter and a mCherry reporter gene at the downstream of the mini promoter, and the upstream of the mini promoter comprises a recombination site of the random sequence.

2. A method of screening a core enhancer using random sequences, the method comprising the steps of:

s1, synthesizing a random sequence library;

s2, carrying out enzyme digestion on the enhancer screening expression vector by using XhOI enzyme and Hind III enzyme to obtain a linear vector;

s3, constructing a plasmid library by using a linear vector and a random sequence library;

s4, transfecting the constructed plasmid library to cells, and based on the fluorescent expression condition, enriching enhancer positive cells through flow;

s5, extracting DNA of the enriched enhancer positive cells, and using the extracted DNA to build a library and then carrying out high-throughput sequencing.

3. The method of claim 2, wherein the random sequence library comprises sequences of 20-50bp in length.

4. The method of claim 2, wherein the random sequence library is diluted before use under the following conditions: 12000g, RT, centrifugation for 90s, adding sterile water to dilute into solution with the concentration of 50 ng/ul.

5. The method for screening a core enhancer using random sequences as claimed in claim 1, wherein the cleavage system in step S2 is: nebuffer2.1, 5 ul; XhoI, 2 ul; hind III, 2 ul; DNA, 20ug, add double distilled water to a system of 50 ul.

6. The method of claim 4, wherein the step S3 specifically comprises:

homologous recombination is carried out on the linear vector and the random sequence library by utilizing a homologous recombinase, recombinant plasmids are added into competent cells, and then the recombinant plasmids are extracted by utilizing a plasmid extraction kit.

7. The method of claim 5, wherein the step S4 specifically comprises:

taking out the cell 293T from the liquid nitrogen, placing in the air for 10-15s, quickly thawing in a 37 ℃ water bath, transferring the cell suspension into a centrifuge tube, centrifuging for 5min at 200g and RT; discarding the supernatant, adding the complete culture medium to suspend the cells, and then placing the cells in an incubator;

when the cells grow to 80-90% confluence, transfecting the cells by using a liposome reagent, and observing the fluorescent expression condition after 48 hours, wherein 2ml of digestive enzyme is added into each 100mm culture dish for digesting the cells for 2min, 2ml of complete culture medium is added for terminating digestion, 200g, RT and centrifugation are carried out for 5 min; discarding the supernatant, adding 5ml of DPBS for washing once, and centrifuging again to collect cells; enrichment of enhancer positive cells using flow sorting cytometry.

8. The method of claim 6, wherein the step S5 of extracting DNA from the enriched enhancer-positive cells comprises:

centrifuging the enriched enhancer positive cells for 5min at 2000g and RT, and discarding the supernatant; adding 600ul of cell lysate, 70ul of 10% SDS, 5ul of proteinase K into the cell sediment, slightly inverting upside down, mixing uniformly, placing in an oven at 56 ℃ for incubation for 1h, and continuously slightly oscillating to digest the cell sediment completely;

adding isovolumetric balance phenol, slightly reversing the upper part and the lower part, mixing uniformly, performing centrifugation for 10min at 13000g, and sucking the supernatant into a new EP tube;

adding equal volume of chloroform: the isoamyl alcohol mixed solution is mixed by slightly reversing the upper part and the lower part, 13000g and RT are centrifuged for 10min, and supernatant is sucked into a new EP tube;

adding two times volume of frozen absolute ethanol at-20 deg.C into the supernatant, precipitating at-20 deg.C for at least 1h, 13000g, RT, centrifuging for 15min, and removing supernatant;

adding 500 μ l of 75% glacial ethanol, mixing, rinsing to remove residual salt, 13000g, RT, centrifuging for 2min, and removing supernatant;

inverting the tube on a paper roll, air-drying the DNA precipitate block, and removing residual ethanol;

adding a proper amount of TE buffer solution or sterilized double distilled water to dissolve the DNA precipitate, and storing for electrophoretic detection.

9. The method of claim 7, wherein the step of constructing a library using the extracted DNA at step S5 comprises:

designing an amplification primer according to the vector, amplifying genome DNA by using high-efficiency amplification enzyme and a PCR amplification program, and then cutting gel, recovering and purifying a target band; and amplifying the gel recovery product in the first step by using a library building kit, and adding a joint for sequencing to the amplified product.

10. The method for screening a core enhancer using random sequences as claimed in claim 8, wherein the PCR amplification process for amplifying genomic DNA using designed amplification primers comprises: 45s at 98 ℃, 10s at 60 ℃, 5s at 72 ℃ and 1min at 72 ℃ for 7 cycles.

11. The method for screening a core enhancer using a random sequence according to claim 8, wherein the products amplified using the pooling kit are ligated by adding a linker for sequencing, 1.5 times by volume of DNA CleanBeads are added to the ligated products, and they are mixed by pipetting 10 times and incubated at room temperature for 5 min;

centrifuging the reaction tube for a short time, placing the reaction tube in a magnetic frame, standing the reaction tube until the solution is clear, and removing the supernatant;

keeping the EP tube in a magnetic frame, adding 200 μ l of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, removing the supernatant, and repeating the steps once;

keeping the EP tube in a magnetic frame, opening the cover and drying the magnetic beads in air for 3-5 min;

taking the EP tube out of the magnetic frame, adding sterilized ultrapure water with a corresponding volume for elution, carrying out vortex oscillation or gently blowing and beating by using a pipettor to fully mix uniformly, standing at room temperature for 2min, centrifuging the reaction tube for a short time, placing in the magnetic frame for standing, and sucking a supernatant into a new EP tube for later use after the solution is clarified;

amplifying the purified product of the DNA Clean Beads by using a KAPA Library amplification Primer Mix, carrying out agarose electrophoresis, then carrying out gel cutting and recovery, and carrying out high-throughput sequencing on the recovered product after detecting the fluorescence concentration.

Technical Field

The invention relates to the field of bioengineering, in particular to an enhancer screening expression vector and a method for screening an enhancer by using a random sequence.

Background

Eukaryotic gene expression is a delicate, complex and dynamically changing process that is subject to complex regulation by multiple factors, such as genomic transcription factors, epigenetic modifications of various regulatory elements, etc., to perform spatio-temporal specific expression of various genes. The cis-regulatory element changes the high-level structure of chromatin by recruiting different trans-acting factors, so as to start or silence the expression of genes, which is a popular field of gene regulation research in recent years, therefore, the identification of the cis-regulatory element in genome is of great significance for deeply understanding the mechanism of gene expression regulation.

Enhancers are cis-regulatory elements with important roles, and have important roles in the regulation of gene expression. The description of genomic features of enhancers is an important part of the encyclopedia project (ENCODE) for DNA elements. For this reason, many methods have been developed, such as chromatin immunoprecipitation (chip-seq), DNase i high sensitive site sequencing (DNase-seq), formaldehyde assisted segregation regulatory element technology, chromatin open sequencing technology (ATAC-seq), and star-seq (self-transcription active regulatory region sequencing), which are mainly chromatin characteristics depending on histone modification or chromatin availability and are considered to be essential characteristics of enhancer sites in general, but such identification of enhancers in genomes is not very accurate due to complexity of chromatin characteristics and analytical methods.

Currently, enhancer elements have been defined mostly over a few hundred base pairs (bp), e.g., H3K27acc chip-seq with a coverage in the range of 200-60Kbp (as a newly defined super enhancer), and such a large region may contain multiple signals. Meanwhile, through motif analysis of these different genomic datasets, we found that the core region of each site is only a few or a few tens of bp in length. It is well known that the identification of the core enhancer in the genome is a nearly impossible task with the existing techniques because these conventional methods cannot achieve a sequence shorter than 147bp due to the structural characteristics of the nucleosomes themselves in the chromosomes.

Therefore, the prior art is subject to further development.

Disclosure of Invention

The present invention has been made in view of the above problems, and an object of the present invention is to provide an enhancer-screening expression vector and a method for screening an enhancer using a random sequence.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an enhancer screening expression vector is used for screening an active enhancer in a random sequence, and is formed by modifying a shuttle plasmid vector, wherein a core component on the enhancer screening expression vector comprises a mini promoter and a mCherry reporter gene at the downstream of the mini promoter, and the upstream of the mini promoter comprises a recombination site of the random sequence.

A method of screening a core enhancer using random sequences, wherein the method comprises the steps of:

s1, synthesizing a random sequence library;

s2, carrying out enzyme digestion on the enhancer screening expression vector by using XhOI enzyme and Hind III enzyme to obtain a linear vector.

S3, constructing a plasmid library by using a linear vector and a random sequence library;

s4, transfecting the constructed plasmid library to cells, and based on the fluorescent expression condition, enriching enhancer positive cells through flow;

s5, extracting DNA of the enriched enhancer positive cells, establishing a library by using the extracted DNA, performing high-throughput sequencing,

the method for screening the core enhancer by using the random sequence is characterized in that the sequence in the random sequence library is 20-50bp in length.

The method for screening the core enhancer by using the random sequence comprises the following steps of diluting a random sequence library before use, wherein the specific dilution conditions are as follows: 12000g, RT, centrifugation for 90s, adding sterile water to dilute into solution with the concentration of 50 ng/ul.

The method for screening the core enhancer by using the random sequence is characterized in that an enzyme cutting system in the step S2 is as follows: nebuffer2.1, 5 ul; XhoI, 2 ul; hind III, 2 ul; DNA, 20ug, add double distilled water to a system of 50 ul.

The method for screening a core enhancer by using a random sequence, wherein the step S3 specifically includes: homologous recombination is carried out on the linear vector and the random sequence library by utilizing a homologous recombinase, recombinant plasmids are added into competent cells, and then the recombinant plasmids are extracted by utilizing a plasmid extraction kit.

The method for screening a core enhancer by using a random sequence, wherein the step S4 specifically includes:

taking out the cell 293T from the liquid nitrogen, placing in the air for 10-15s, quickly thawing in a 37 ℃ water bath, transferring the cell suspension into a centrifuge tube, centrifuging for 5min at 200g and RT; discarding the supernatant, adding the complete culture medium to suspend the cells, and then placing the cells in an incubator;

when the cells grow to 80-90% confluence, transfecting the cells by using a liposome reagent, and observing the fluorescent expression condition after 48 hours, wherein 2ml of digestive enzyme is added into each 100mm culture dish for digesting the cells for 2min, 2ml of complete culture medium is added for terminating digestion, 200g, RT and centrifugation are carried out for 5 min; discarding the supernatant, adding 5ml of DPBS for washing once, and centrifuging again to collect cells; enrichment of enhancer positive cells using flow sorting cytometry.

The method for screening a core enhancer by using a random sequence, wherein the step S5 of extracting DNA from the enriched enhancer positive cells specifically comprises the following steps:

centrifuging the enriched enhancer positive cells for 5min at 2000g and RT, and discarding the supernatant; adding 600ul of cell lysate, 70ul of 10% SDS, 5ul of proteinase K into the cell sediment, slightly inverting upside down, mixing uniformly, placing in an oven at 56 ℃ for incubation for 1h, and continuously slightly oscillating to digest the cell sediment completely;

adding isovolumetric balance phenol, slightly reversing the upper part and the lower part, mixing uniformly, performing centrifugation for 10min at 13000g, and sucking the supernatant into a new EP tube;

adding equal volume of chloroform: the isoamyl alcohol mixed solution is mixed by slightly reversing the upper part and the lower part, 13000g and RT are centrifuged for 10min, and supernatant is sucked into a new EP tube;

adding two times volume of frozen absolute ethanol at-20 deg.C into the supernatant, precipitating at-20 deg.C for at least 1h, 13000g, RT, centrifuging for 15min, and removing supernatant;

adding 500 μ l of 75% glacial ethanol, mixing, rinsing to remove residual salt, 13000g, RT, centrifuging for 2min, and removing supernatant;

inverting the tube on a paper roll, air-drying the DNA precipitate block, and removing residual ethanol;

adding a proper amount of TE buffer solution or sterilized double distilled water to dissolve the DNA precipitate, and storing for electrophoretic detection.

The method for screening a core enhancer by using a random sequence, wherein the step S5 of creating a library by using the extracted DNA specifically includes:

designing an amplification primer according to the vector, amplifying genome DNA by using high-efficiency amplification enzyme and a PCR amplification program, and then cutting gel, recovering and purifying a target band; and amplifying the gel recovery product in the first step by using a library building kit, and adding a joint for sequencing to the amplified product.

The method for screening the core enhancer by using the random sequence is characterized in that the PCR amplification program for amplifying the genomic DNA by using the designed amplification primers is as follows: 45s at 98 ℃, 10s at 60 ℃, 5s at 72 ℃ and 1min at 72 ℃ for 7 cycles.

The method for screening the core enhancer by using the random sequence comprises the steps of adding a sequencing adaptor to a product amplified by using a library building kit for connection, adding 1.5-time volume of DNA Clean Beads to the connected product, blowing and beating the product by using a liquid moving machine for 10 times, uniformly mixing the product and the DNA Clean Beads, and incubating the product at room temperature for 5 min;

centrifuging the reaction tube for a short time, placing the reaction tube in a magnetic frame, standing the reaction tube until the solution is clear, and removing the supernatant;

keeping the EP tube in a magnetic frame, adding 200 μ l of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, removing the supernatant, and repeating the steps once;

keeping the EP tube in a magnetic frame, opening the cover and drying the magnetic beads in air for 3-5 min;

taking the EP tube out of the magnetic frame, adding sterilized ultrapure water with a corresponding volume for elution, carrying out vortex oscillation or gently blowing and beating by using a pipettor to fully mix uniformly, standing at room temperature for 2min, centrifuging the reaction tube for a short time, placing in the magnetic frame for standing, and sucking a supernatant into a new EP tube for later use after the solution is clarified;

amplifying the purified product of the DNA Clean Beads by using a KAPA Library amplification Primer Mix, carrying out agarose electrophoresis, then carrying out gel cutting and recovery, and carrying out high-throughput sequencing on the recovered product after detecting the fluorescence concentration.

Has the advantages that:

the invention provides an enhancer screening expression vector and a method for screening an enhancer by using a random sequence, wherein the method utilizes the enhancer to screen the expression vector and screen a library to realize effective identification of an enhancer sequence.

Drawings

FIG. 1 is a flow chart of the experimental principle of the present invention;

FIG. 2 is a map of an enhancer screening expression vector in an embodiment of the present invention;

FIG. 3 is a diagram of agarose gel electrophoresis of an enhancer screening expression vector after digestion with XhOI and Hind III in an embodiment of the present invention;

FIG. 4 is a diagram of the electrophoresis of the homologous recombinant plasmid pmx-mp-N25-mcherry agarose gel in the embodiment of the present invention;

FIG. 5 is a fluorescence image of cells transfected with the random sequence library of the present invention.

FIG. 6 shows the enrichment results of the cell flow type screening of the present invention.

FIG. 7 is a two-step library construction sequencing agarose gel electrophoresis chart of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived from the embodiments of the present invention by a person skilled in the art without any creative effort, should be included in the protection scope of the present invention.

As shown in figure 1, the invention carries out homologous recombination on a random synthetic sequence (20-50bp) to the upstream of a mini promoter, activates the expression of a downstream reporter gene mChery if the random sequence has enhancer activity, screens enhancer positive cells through flow enrichment, and obtains an enhancer sequence through library construction and high-throughput sequencing.

The enhancer screening expression vector is used for screening an active enhancer in a random sequence, the enhancer screening expression vector is formed by modifying a shuttle plasmid vector, a core component on the enhancer screening expression vector comprises a mini promoter and a mcherry reporter gene at the downstream of the mini promoter, the upstream of the mini promoter comprises a recombination site of the random sequence, and a specific map of the enhancer screening expression vector (pmx-mcherry) is shown in figure 2.

The specific experimental process is as follows:

1. synthesis of random sequence library: a25 bp long random sequence library was synthesized by Jinzhi Biotechnology Ltd.

Random library dilution: 12000g, RT, centrifugation for 90s, adding sterile water to dilute into solution with the concentration of 50 ng/ul.

2. Vector linearization

2.1 vector double digestion

Using XhOI (NEB.Cat.No. R0146V) and Hind III (NEB.Cat.No. R0104b) at NEBuffer2.1(NEB.Cat.No. B7202S)

The pmx-mcherry vector (FIG. 2) was digested in buffer, and the sequence electrophoresis after digestion is shown in FIG. 3, and the digestion system is as follows:

components	Dosage of
		NEBuffer2.1	5ul
XhoI	2ul
		Hind III	2ul
DNA	20ug
		dd water	To 50ul

2.2 recovery of the digestion vector glue

Weighing 0.5g of rubber powder, dissolving in 50ul 1 XTAE, heating in a microwave oven for 1min, taking out, shaking for several times, heating for 1min, pouring into a rubber plate, and standing for 30 min. Taking supercoiled DNA Ladder (NEB; Cat. No. N0472S) as a control, setting parameters of 120V, 130mA and 45min for agarose electrophoresis at the loading amount of 200 ng/well and 500 ng/well; the band of interest was recovered with a gel recovery kit (Zymo; Cat. No. D4008).

Plasmid library construction

3.1 construction of homologous recombination plasmid (pmx-mp-N25-mcherry)

Homologous recombination is carried out on linear vectors and random libraries by using homologous recombinase (Vazyme; Cat. No. C112), each reaction is carried out for 10ng of random library, 120ng of linear vectors and 37 ℃ for 30min, recombination products are added into competent cells (TSINGKE; Cat. No. TSC01), the mixture is gently mixed, the mixture is stood on ice for 30min, 42 ℃ water bath 45S, immediately taken out of the ice and stood for 2-3min, 500ul of SOC culture medium is added into each tube to be activated in a shaker at 37 ℃ and 200rpm for 1h, the mixture is taken out and put into a centrifuge for 2000g, RT and 5min, supernatant is discarded, 100ul of SOC heavy suspension thalli is left, the thalli are evenly coated on a solid culture plate, and the culture box at 37 ℃ is used for overnight culture (the specific time depends on the competence efficiency).

3.2 plasmid extraction

The colonies were scraped off by adding LB medium to the solid dish and the flask was supplemented with the appropriate volume of LB medium (AMP added) setting the shaker parameters 37 ℃ and shaking at 200rpm for 12-16 hours. The following day, plasmid was extracted using a plasmid extraction kit (OMEGA; Cat. No. D6950), and the plasmid quality was checked by agarose gel electrophoresis (FIG. 4).

Cell culture

Taking out cells (293T) from liquid nitrogen, standing in air for 10-15s, quickly thawing in a 37 deg.C water bath, transferring cell suspension into a centrifuge tube, centrifuging at 200g and RT for 5 min; the supernatant was discarded, and the cells were resuspended in complete medium (90% DMEM (ThermFisher; Cat. No.10566024) + 10% FBS (Gibco; Cat. No.10437028)) and then placed in an incubator.

Cell transfection and enhancer positive cell enrichment

When the cells grew to 80-90% confluence, the cells were transfected with a liposome reagent (promega; E2311) while pmx-mp-mcherry and pmx-SP 40-mp-mcherry were set as negative and positive controls, respectively. After 48h, observing the fluorescence expression (figure 5), adding 2ml of digestive enzyme (Thermofisher; Cat. No. A1110501) into each 100mm culture dish for digesting the cells for 2min, adding 2ml of complete culture medium to terminate digestion, and centrifuging for 5min at 200g and RT; discarding the supernatant, adding 5ml of DPBS for washing once, and centrifuging again to collect cells; enhancer positive cells were enriched using flow sort cytometry (figure 6).

Sequencing and library building

6.1 extraction of genomic DNA from Positive cells

Centrifuging the enriched cells for 5min at 2000g and RT, and removing the supernatant; adding 600ul of cell lysate (STE), 70ul of 10% SDS (final concentration of 1%), 5ul of proteinase K (final concentration of 100-200 ug/ml), slightly inverting and reversing the number of the cells, mixing, incubating in an oven at 56 ℃ for 1h, and continuously and slightly shaking to digest the cells completely.

An extraction step 1: add an equal volume of equilibrated phenol (about 700. mu.l), mix gently upside down, 13000g, centrifuge for 10min, carefully pipette the supernatant into a new EP tube.

An extraction step 2: adding equal volume of chloroform: isoamyl alcohol (24: 1) mixture, mix gently upside down, 13000g, RT, centrifuge for 10min, carefully aspirate the supernatant into a new EP tube. If a white protein layer is arranged in the middle, the extraction steps 1 and 2 are repeated until no white protein layer exists.

And (3) precipitation: adding two times volume of frozen anhydrous ethanol at-20 deg.C into the supernatant, precipitating at-20 deg.C for at least 1h, 13000g, RT, centrifuging for 15min, and discarding the supernatant. Note that this step requires careful handling to prevent the DNA pellet from being discarded altogether.

Rinsing: add 500. mu.l of 75% glacial ethanol, rinse well to remove residual salts, 13000g, RT, centrifuge for 2min, and aspirate supernatant. If the impurities are more, the procedure can be repeated several times.

And (3) drying: the tube was inverted on a paper roll, and the DNA pellet was air dried to remove residual ethanol.

Dissolving: adding appropriate amount of TE (pH 8.0) or sterilized double distilled water to dissolve DNA precipitate, and storing for electrophoresis detection.

6.2 building a library

The library construction method is divided into two steps of PCR library construction.

The first step is as follows: the genomic DNA was amplified (7 cycles) using high efficiency amplification enzymes (KAPA; Cat. No. KK2601) and PCR amplification protocols (98 ℃ for 45s, 98 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 5s, 72 ℃ for 1min) using vector-designed amplification primers (this experiment: pMX-TY1:5 '-TGCAGGTGCCAGAACATTTC-3' pMX-TY2:5 '-GTGGCTTTACCAACAGTACC-3'), and then subjected to gel cutting to recover the purified band of interest (FIG. 7).

The second step is that: the recovered product of the first step is subjected to EndRepiair and A-tailing by a kit for building a library (KAPA; Cat.No. KK8504) in the following system:

amplification was performed according to the following procedure:

the amplified products were ligated with a linker for sequencing (Vazyme; Cat. No. N805) as follows:

the above system was reacted in a PCR instrument at 20 ℃ for 15 minutes.

Adding 1.5 times volume of DNA Clean Beads (beckman; Cat.No. A63881) into the mixed system, beating with a pipette for 10 times, mixing, and incubating at room temperature for 5 min;

the reaction tube was centrifuged briefly and placed in a magnetic stand to stand until the solution cleared (about 5min), and the supernatant was removed.

The EP tube was kept in the magnetic rack and 200. mu.l of freshly prepared 80% ethanol was added to rinse the beads. Incubate at room temperature for 30s, remove the supernatant, repeat this step once.

Keeping the EP tube in the magnetic frame, uncovering the lid and air-drying the magnetic beads for 3-5min (the beads cannot be chapped).

The EP tube was removed from the magnetic stand and eluted with a corresponding volume of sterile ultrapure water. Vortex and shake or gently blow and beat by using a pipette, and fully mix, and place for 2min at room temperature. The reaction tube was centrifuged briefly and placed in a magnetic stand to stand until the solution cleared (about 5min), and the supernatant carefully pipetted into a new EP tube for use.

The products after the purification of Beads were amplified for 4 cycles with KAPA Library amplification Primer Mix according to the amplification procedure of the first step, subjected to 120V, 130mA agarose electrophoresis for 35min, then to gel cutting recovery (FIG. 7), and subjected to high throughput sequencing after the concentration detection of the recovered product, namely the Qubit.

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.