CN118652958A - A DNA affinity purification sequencing library construction method based on double pull down - Google Patents

Abstract

The present application discloses a DNA affinity purification sequencing library construction method based on double pull down, which belongs to the field of gene sequencing technology. The method provided in the present application is that after two different proteins bind to each other in vitro, they further bind to DNA to obtain DNA bound to the two protein complexes, and then perform high-throughput sequencing. Compared with ChIP-seq, this method can solve the problems of antibody limitation, low repeatability success rate, and the need for toxic reagents; compared with ordinary DAP-seq, it can detect binding sites for transcription factors that form heterodimers, and can also detect the interaction between two transcription factors.

Description

A DNA affinity purification sequencing library construction method based on double pull down

Technical Field

The present application belongs to the field of gene sequencing technology, and in particular relates to a DNA affinity purification sequencing library construction method based on double pull down.

Background Art

ChIP-seq is one of the important methods for studying epigenetics. It can effectively detect transcription factor binding sites when the antibody quality is good. However, its limitations are as follows: in vivo experiments have low repeatability and success rate; toxic reagents such as formaldehyde are required; and the antibody requirements are very high, making it difficult to conduct experiments on all transcription factors.

Based on the problems of ChIP-seq, a method for in vitro transcription factor-DNA interaction was derived, DNA affinity purification sequencing, or DAP-seq. DAP-seq solves the instability of in vivo experiments and the high requirements for antibodies in experiments, and conducts in vitro experiments without the need for high-quality antibodies for transcription factors.

However, the current limitation of DAP-seq is that it can only perform affinity purification sequencing of single transcription factor monomers and DNA, and cannot resolve the detection of binding sites of homologous, heterologous dimers or polymers with DNA.

Summary of the invention

To solve the above technical problems, the present application provides a DNA affinity purification sequencing library construction method based on double pull down. In this method, two different proteins bind to each other in vitro and then further bind to DNA to obtain DNA bound to the two protein complexes, which is then subjected to high-throughput sequencing.

In order to achieve the above-mentioned invention object, this application provides the following technical solutions:

The present application provides a method for DNA affinity purification sequencing library construction based on double pull down, comprising the following steps:

(1) Extracting genomic DNA from target samples;

(2) fragmenting the genomic DNA to obtain DNA fragments;

(3) constructing a library of the DNA fragments to obtain a DNA library;

(4) obtaining two transcription factor expression vectors with different tags;

(5) placing equal amounts of the two transcription factor expression vectors in the same in vitro protein expression system, performing in vitro protein expression, and obtaining expression products;

(6) using different tag protein purification magnetic beads to purify the expression products in sequence to obtain purified products;

(7) combining the purified product with the DNA library in step (3), incubating and eluting in sequence to obtain an eluted DNA library;

(8) performing PCR amplification on the eluted DNA library to obtain a sequencing library;

(9) Sequencing and analyzing the sequencing library.

Optionally, in step (2), the size of the DNA fragment is 200 to 400 bp.

Optionally, in step (2), the size of the DNA fragment is independently selected from any value among 200 bp, 250 bp, 300 bp, 350 bp, 400 bp or any range between two of them.

Optionally, in step (3), the library construction sequentially includes end repair and linker ligation.

Optionally, the end repair reaction system is: 0.8-1.2 μL of DNA fragment, 2.8-3.2 μL of NEXTflex ^TM End-Repair&Adenylation Enzyme Mix 2.0, 2.8-3.2 μL of NEXTflex ^TM End-Repair&Adenylation Buffer Mix 2.0, and NFW is added to 50 μL.

Preferably, the end repair reaction system is: 1 μL of DNA fragment, 3 μL of NEXTflex ^TM End-Repair & Adenylation Enzyme Mix 2.0, 3 μL of NEXTflex ^TM End-Repair & Adenylation Buffer Mix 2.0, and NFW is added to 50 μL.

Optionally, the reaction conditions of the end repair are: 18-22°C, 28-32min→64-66°C, 28-32min→3-5°C, constant temperature incubation.

Preferably, the reaction conditions of the end repair are: 20°C, 30 min→65°C, 30 min→4°C, constant temperature incubation.

Optionally, the reaction system for the linker connection is: 48-52 μL of end repair product, 2.8-3.2 μL of genobaits linker, 44-45 μL of NEXTflex ^TM Ligase Enzyme Mix, and 2.8-3.2 μL of NEXTflex ^TM Ligase Buffer Mix.

Preferably, the reaction system for the linker ligation is: 50 μL of end repair product, 3 μL of genobaits linker, 44.5 μL of NEXTflex ^TM Ligase Enzyme Mix, and 3 μL of NEXTflex ^TM Ligase Buffer Mix.

Optionally, the reaction conditions for the linker connection are: 18-22°C, 14-16min→3-5°C, constant temperature incubation.

Preferably, the reaction conditions for the linker connection are: 20°C, 15min→4°C, constant temperature incubation.

Optionally, step (4) includes:

S1, select transcription factor 1 and transcription factor 2;

S2, design PCR amplification primers according to the coding CDS sequences of transcription factor 1 and transcription factor 2 respectively;

S3, using PCR amplification primers to amplify the coding CDS sequence of transcription factor 1 and the coding CDS sequence of transcription factor 2 respectively;

S4, constructing the CDS sequence encoding transcription factor 1 and the CDS sequence encoding transcription factor 2 into protein expression vector 1 and protein expression vector 2 respectively;

S5, transforming the protein expression vector 1 and the protein expression vector 2 into Escherichia coli for cultivation respectively;

S6, picking up single clone colonies for sequencing, screening out expression vectors that are consistent with the coding CDS sequences of transcription factor 1 and transcription factor 2, and obtaining expression vector strains;

S7, extracting the protein expression plasmid of transcription factor 1 and the protein expression plasmid of transcription factor 2, thus obtaining two transcription factor expression vectors with different tags;

In step S1, there is an interaction relationship between the transcription factor 1 and the transcription factor 2, and they can jointly regulate the expression of the same gene.

Optionally, in step S2, the coding CDS sequence of transcription factor 1 is as shown in SEQ ID No.1.

Optionally, the coding CDS sequence of transcription factor 2 is shown as SEQ ID No.2.

Optionally, in step S3, the amplification primers for the CDS sequence encoding transcription factor 1 are shown as SEQ ID No. 3 and SEQ ID No. 4.

Optionally, primers for amplifying the CDS sequence encoding transcription factor 2 are shown as SEQ ID No.5 and SEQ ID No.6.

Optionally, in step S4, the initial expression vectors of the protein expression vector 1 and the protein expression vector 2 are independently pf3k.

Optionally, the protein expression vector 1 carries a Flag tag;

The upstream sequence of the Flag tag is shown in SEQ ID No.7;

The downstream sequence of the Flag tag is shown as SEQ ID No.8.

Optionally, the protein expression vector 2 carries a Myc tag;

The upstream sequence of the Myc tag is shown in SEQ ID No.9;

The downstream sequence of the Myc tag is shown as SEQ ID No.10.

Optionally, in step (5), the reagents for in vitro protein expression include: 28-32 μL of TNT mix, 0.8-1.2 μL of each of two transcription factor expression vectors, and enzyme-free water to make up to 50 μL.

Preferably, in step (5), the reagents for in vitro protein expression include: 30 μL of TNT mix, 1 μL of each of two transcription factor expression vectors, and enzyme-free water to make up to 50 μL.

Optionally, the temperature for in vitro protein expression is 28-32°C;

The in vitro protein expression time is 1.8 to 2.2 hours.

Optionally, the temperature for the in vitro protein expression is independently selected from any value among 28°C, 29°C, 30°C, 31°C, 32°C, or any range therebetween.

Optionally, the time of in vitro protein expression is independently selected from any value among 1.8h, 1.9h, 2h, 2.1h, 2.2h, or a range between any two of them.

Optionally, in step (6), the different-tagged protein purification magnetic beads include Flag tag purification magnetic beads and Myc tag purification magnetic beads.

Optionally, in step (7), the incubation time is 1.8 to 2.2 hours.

Optionally, in step (7), the incubation time is independently selected from any value among 1.8h, 1.9h, 2h, 2.1h, 2.2h, or any range between two of them.

Optionally, in step (8), the PCR amplification reaction system includes: 14-16 μL of eluted DNA library, 0.8-1.2 μL of Universal Primer, 0.8-1.2 μL of Index Primer, 24-26 μL of 2×KAPA HIFImix, and enzyme-free water to make up to 50 μL.

Preferably, in step (8), the PCR amplification reaction system comprises: 15 μL of eluted DNA library, 1 μL of Universal Primer, 1 μL of Index Primer, 25 μL of 2×KAPA HIFI mix, and enzyme-free water to make up to 50 μL.

Optionally, the nucleotide sequence of the Universal Primer is shown in SEQ ID No.11;

The nucleotide sequence of the Index Primer is shown in SEQ ID No.12.

Optionally, in step (8), the reaction procedure of the PCR amplification is: 97-99°C, 2.5-3.5min, 1cycle→97-99°C, 9-11s; 58-62°C, 28-32s; 70-73°C, 28-32s; 28-32cycle→70-73°C, 1.5-2.5min, 1cycle→3-5°C, constant temperature for standby use.

Preferably, in step (8), the reaction procedure of the PCR amplification is: 98°C, 3min, 1cycle→98°C, 10s; 60°C, 30s; 72°C, 30s; 30cycle→72°C, 2min, 1cycle→4°C, constant temperature for standby use.

Optionally, in step (9), the sequencing strategy is PE150, and the sequencing volume is 5.8 to 6.2G.

Optionally, in step (9), the sequencing amount is independently selected from any value among 5.8G, 5.9G, 6G, 6.1G, 6.2G, or a range between any two of them.

Compared with the prior art, this application has the following beneficial effects:

The method provided in this application is that after two different proteins bind to each other in vitro, they further bind to DNA to obtain DNA bound to the two protein complexes, and then perform high-throughput sequencing. Compared with ChIP-seq, this method can solve the problems of antibody limitation, low repeatability success rate, and the need for toxic reagents; compared with ordinary DAP-seq, it can detect binding sites of transcription factors that form heterodimers, and can also detect the interaction between two transcription factors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

FIG1 is a plasmid information diagram of protein expression vector 1 (pf3k-flag) and protein expression vector 2 (pf3k-Myc) of the present application;

FIG2 is the DNA extraction result of the present application;

FIG3 is the protein pre-expression result of the present application;

FIG4 is the library construction result of this application.

DETAILED DESCRIPTION

The present application is further described below in conjunction with specific embodiments. The following are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application discloses the following preferred embodiments, they are not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

Example 1

In this example, WRKY and HSF transcription factors from poplar were selected for experiment.

WRKY is named for its special heptapeptide conserved sequence WRKYGOK. It is one of the largest transcription factor families in plants and is widely involved in the response of plants to biotic, abiotic and hormonal stresses. WRKY TFs mainly regulate the expression of target genes by specifically binding to the cis-acting elements (W-box) of the target gene promoter and participate in the signaling pathways of plant responses to different biotic, abiotic and hormonal stresses, as well as interacting with multiple proteins to achieve their functions in different signal transduction pathways. The HSF transcription factor family is one of the key components of plant heat stress response and plays an important role in plant heat stress memory. As an environmental adaptive response, plants can acquire heat tolerance under moderate heat stress conditions. This heat tolerance can be maintained for a period of time and the process involves the continuous induction of memory-related genes, thereby improving their tolerance to subsequent heat stress.

The two transcription factors selected in this example can interact with each other to regulate the expression of the target gene.

1. Primer Design

PCR amplification primers were designed according to the coding CDS (Coding sequence) sequences of transcription factors WRKY and HSF, wherein the coding CDS sequence of WRKY is shown in SEQ ID No.1, and the coding CDS sequence of HSF is shown in SEQ ID No.2.

The vector was constructed using homologous recombination. The initial vector was pf3k. The upstream and downstream sequences of the protein expression vector insertion site were as follows:

The upstream sequence of the Flag tag vector is shown in SEQ ID No.7: ATTGCCTGACAAGCTGAGGGCCACCCTTCTATCCCCACCGCGCGATC GCG

The downstream sequence is shown in SEQ ID No. 8: GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAA.

The upstream sequence of the Myc tag vector is shown in SEQ ID No. 9: ATTGCCTGACAAGCTGAGGGCCACCCTTCTATCCCCACCGCGCGATC GCG;

The downstream sequence is shown in SEQ ID No. 10: GAGCAGAAACTCATCTCTGAAGAGGATCTGGAGCAGAAACTCATCT CTGA.

The plasmid information diagram of the final protein expression vector 1 (pf3k-flag) and protein expression vector 2 (pf3k-Myc) is shown in FIG1 .

The amplification primers of WRKY transcription factor and HSF transcription factor were designed based on the gene sequences, as shown in Table 1.

Table 1

2. Amplification of transcription factor target fragments

1. The amplification template contains the plasmid of the transcription factor target gene. The concentration is quantified using Qubit and then diluted. The final concentration of the dilution is ~1ng/μl. The amount of template input for amplification is 1ng.

2. Enzymes and buffers must be placed on ice immediately after being taken out of the refrigerator. Other reagents can be placed on a double-sided plate to be slowed down at room temperature.

3. Prepare the target fragment PCR amplification reaction system and place it on ice;

4. After configuring the system, flick or vortex to mix, centrifuge instantly to ensure that all the reaction solution is at the bottom of the PCR tube, there is no liquid residue on the side wall, and there are no bubbles in the tube (a small amount of bubbles is acceptable).

5. Perform PCR amplification.

6. After amplification, perform 1.2% agarose gel electrophoresis on the PCR product. Using a scalpel blade, cut out the gel containing the DNA fragment of the expected size against the marker.

7. Use the Adlay Gel Recovery Kit to recover the gel.

3. Seamless cloning

The cloning ligation was performed using the Novagen Seamless Cloning Kit C112.

Configure the system according to the kit instructions:

Linearized vector 80ng

DNA target fragment 60ng

5×CE buffer 2μl

Recombinase 1 μl

ddH ₂ O to 10 μl

After adding the system, flick or vortex to mix, centrifuge instantly to ensure that all the reaction solution is at the bottom of the PCR tube, there is no liquid residue on the side wall, and there are no bubbles in the tube (a small amount of bubbles is acceptable), and place it on the PCR instrument at 37℃ for 30 minutes.

4. Plasmid Transformation

Take the competent cells out of the -80°C freezer, quickly put them into ice, and thaw them in the ice.

Add 5 μl of ligation reaction solution to the competent cell suspension and gently rotate the centrifuge tube to mix the contents. After mixing, place the tube on ice for 30 minutes.

Place the centrifuge tube in a 42°C metal bath for 45 seconds, then quickly transfer the tube to an ice bath to cool the cells for 2 minutes. Do not shake the centrifuge tube during this process.

Under sterile conditions (turn on the ultra-clean workbench for UV sterilization for 15 minutes before use, and ventilate for 5 minutes), add 900 μl of sterile liquid LB medium (without antibiotics) to each centrifuge tube, mix well and place on the float, put in a constant temperature shaker at 37°C 200 rpm, and shake and culture for 1 hour to allow the bacteria to revive.

5. Colony culture

Place the revived bacteria in step 4 in a centrifuge and centrifuge at 3000 rpm for 5 min.

Under sterile conditions, the supernatant was discarded and the precipitate was spread evenly on an LB solid culture medium dish containing the corresponding antibiotic (the resistance of the carrier used was Kan+) using a spreading rod.

After the liquid in the culture dish is completely absorbed, seal it with a sealing film.

Invert the culture dish and incubate at 37°C overnight (16 hours).

VI. Monoclonal colony identification

Under sterile conditions, prepare sterilized 8-strip PCR tubes or 1.5 ml centrifuge, mark the sample name with a marker, and add 10 μl of enzyme-free water.

Take out the culture dish with colonies, remove the sealing film wrapped around the outside, open the lid of the culture dish, aim at the larger single colony, gently pick it out with a 10μl pipette tip, blow and stir in the prepared enzyme-free water, and take 4 monoclonal colonies from each culture dish.

Prepare a new 8-row colony PCR reaction system:

Template (enzyme-free water from the previous step) 1 μl

Tag enzyme (2×) 5 μl

Primer (forward) 1 μl

Primer (reverse) 1 μl

_ddH2O 2μl

After adding the system, flick or vortex to mix, centrifuge instantly to ensure that all the reaction solution is at the bottom of the PCR tube, there is no liquid residue on the side wall, and there are no bubbles in the tube (a small amount of bubbles is acceptable), and then put it on the PCR instrument for amplification.

Add 100 μl of kan-resistant liquid LB to the remaining 9 μl of enzyme-free water, and place in a constant temperature shaker at 37°C and 150 rpm for incubation for 6 hours.

After the PCR was completed, 5 μl of the PCR product was taken for 1.8% agarose electrophoresis.

VII. Bacterial liquid sequencing

According to the colony PCR electrophoresis, bacterial liquid that meets the expected size is selected for first-generation sequencing.

After the sequencing results are returned, DNAMAN software is used to compare the sequencing results with the gene sequence to obtain a successfully sequenced bacterial liquid sample.

8. Plasmid Extraction

After the sequence alignment results are consistent, mix the remaining bacterial solution, take 10 μl and put it into 10 ml LB liquid culture medium containing antibiotics (Kan+), and culture it overnight at 37°C and 200 rpm. It is best if the turbidity is obviously visible to the naked eye.

The plasmid was extracted using the Tiangen High-Purity Plasmid Miniprep Kit.

After the extraction, the plasmid was subjected to Qubit concentration determination and 1.8% agarose electrophoresis detection to obtain the plasmid concentration.

IX. Protein Pre-expression

The protein was expressed using the Promega wheat germ cell-free protein expression system.

Configure the reaction system as follows:

TNT mix 6μl

Protein expression vector Flag or Myc 400ng

Enzyme-free water to 10μl

After the reaction system is mixed, it is placed in a constant temperature metal bath and cultured at 30°C for 2 h.

After the incubation, add 2 μl 5x SDS loading to the expression system, mix well, and incubate at 98°C for 10 min in a constant temperature metal bath to denature the protein for WB detection.

The protein pre-expression results are shown in Figure 2.

10. Pre-expression Wb detection

Prepare SDS-PAGE gel.

The denatured protein was loaded onto the gel and subjected to electrophoresis.

After electrophoresis, the membrane was transferred using the trans-turbo semi-dry membrane transfer apparatus from bio-rad company to transfer the test samples and markers from the PAGE gel to the PVDF membrane.

The PVDF membrane with the sample to be tested is endowed with the primary antibody solution of the corresponding antibody. In this experiment, WRKY and HSF transcription factors correspond to FLAG and MYC tags respectively.

After the primary antibody incubation, rinse with solution.

After rinsing, the secondary antibody incubation was performed. The secondary antibody was selected according to the species of the primary antibody. In this application, the primary antibody was a mouse antibody, and the secondary antibody was a goat anti-mouse antibody.

After the secondary antibody incubation, rinse with solution.

After the secondary antibody was washed, the test was performed using the Melun ECL detection kit.

Use a gel imager to take pictures in chemiluminescence mode to detect whether the size of the protein band to be tested is consistent with expectations.

11. DNA Extraction

Place 2.0 ml centrifuge tubes pre-filled with steel beads on the double-sided board. Prepare at least 4 tubes for each tissue material.

Take out the tissue material from the liquid nitrogen, quickly open the package, use pre-cooled small tweezers to grab the sample, cut the sample into small pieces as quickly as possible, quickly put it into a 2.0ml centrifuge tube pre-filled with steel balls, cover the lid, and immediately put the centrifuge tube into liquid nitrogen.

Immerse the metal module of the automatic grinder in liquid nitrogen for precooling, and then place it on the grinder. Place the 2.0ml centrifuge tube containing the sample into the module and run the grinder. After stopping, take out the sample and quickly observe whether the ground sample meets the standard (no large particles can be seen with the naked eye).

Remove the centrifuge tube, add 900 μl of 2% CTAB solution into the centrifuge tube, and place the centrifuge tube in a metal bath and incubate at 65°C for 40 minutes. During this period, invert or vortex to mix three times.

Take out the centrifuge tube and place it in a desktop centrifuge for centrifugation at 12000rmp for 5 minutes. Prepare a new 2.0ml centrifuge tube during the centrifugation.

Slowly pipette 700 μl of supernatant into a new centrifuge tube prepared in advance, add an equal volume of DNA extraction reagent (Gold Clone EX-0128), mix up and down, and centrifuge in a desktop centrifuge at 12,000 rpm for 10 minutes.

Prepare a new 1.5 ml centrifuge tube during centrifugation, slowly pipette 500 μl of supernatant into the prepared new centrifuge tube, add 350 μl of pre-cooled isopropanol, invert or vortex to mix, and place in a -20°C refrigerator for 1 hour.

Take out the centrifuge tube and put it into a desktop centrifuge for centrifugation at 12000rmp for 5 minutes. Discard the supernatant. Add 600μl of 80% ethanol solution to the precipitate in the centrifuge tube, turn it upside down several times, transfer the same sample precipitate to the same centrifuge tube, discard the alcohol, open the lid and dry the precipitate.

After the precipitate in the tube is completely dried, add 80μl of ddH ₂ O containing RNase, and place the centrifuge tube in a metal bath for incubation at 37°C for 30 minutes. After the DNA is dissolved, take 1μl of DNA for Qubit quantification, and take 50ng for agarose electrophoresis according to the concentration. The DNA extraction results are shown in Figure 1.

12. Library Construction

The DNA library was constructed using the Bioo Scientific DNA library construction kit, catalog number 5188-03.

Take 1 μg of the extracted genomic DNA and use the covaris s220 instrument to fragment the DNA. The expected size of the DNA fragment is 200-400 bp, and the main band is around 300 bp.

Prepare the end repair reagents according to the reaction system in Table 2:

Table 2

Mix well by pipetting, collect by instant centrifugation, and place in a PCR instrument for incubation. The reaction conditions are shown in Table 3:

Table 3

According to the reaction system in Table 4, configure the adapter ligation reagents:

Table 4

Mix well by pipetting, collect by instant centrifugation, and place in a PCR instrument for incubation. The reaction conditions are shown in Table 5:

Table 5

After the connection is completed, add 65 μl of enzyme-free water and 35 μl of Cleanup Beads2.0, mix thoroughly, and let stand at room temperature for 5 minutes.

Place the centrifuge tube on a magnetic rack, wait for the solution to become clear, discard the supernatant, add 80% ethanol, and rinse twice.

Discard all ethanol and place the centrifuge tube on the magnetic rack to dry until the surface of the magnetic beads is matte.

Add 35 μl of enzyme-free water to resuspend the magnetic beads, mix thoroughly, and let stand at room temperature for 5 minutes.

Place the centrifuge tube on a magnetic stand, wait for the solution to become clear, and transfer the supernatant to a new centrifuge tube, which is the DNA library. The library construction result is shown in FIG3 .

13. DNA library quality inspection

Take 1 μl of the library obtained in step 12 and dilute it 40,000 times, and store the remaining library in a -20°C refrigerator.

According to the reaction system in Table 6, configure the quality inspection reagents:

Table 6

The reaction procedure is shown in Table 7:

Table 7

14. Protein Expression

According to the reaction system in Table 8, the protein expression reagents were prepared and two expression vectors were used for protein expression in the same reaction system:

Table 8

After the reaction system is mixed, it is placed in a constant temperature metal bath and cultured at 30°C for 2 h.

After the incubation, take out 10 μl into a new EP tube, add 2 μl 5x SDS loading, mix well, and incubate at 98°C for 10 min in a constant temperature metal bath to denature the protein for WB detection.

The remaining 40 μl of protein was placed in a 4°C refrigerator for later use.

15. Protein Purification

1. Use Myc and Flag tag protein purification magnetic beads to purify the corresponding tag proteins. The products used are Flag tag purification magnetic beads (sigma-ANTI-FLAG M2 Magnetic Beads) and Myc tag purification magnetic beads (Thermo-Pierce anti-c-Myc magnetic beads).

2. Take the labeled magnetic beads out of the refrigerator, mix them at room temperature, and take out 20 μl into a new 1.5 ml EP tube.

3. Place the centrifuge tube on a magnetic rack for adsorption. After the solution becomes clear, discard the supernatant.

4. Take out the centrifuge tube, add 100 μl IP Buffer to resuspend the magnetic beads, and mix well by pipetting.

5. Place the centrifuge tube on a magnetic rack for adsorption. After the solution becomes clear, discard the supernatant.

6. Repeat steps 3-4 for a total of 3 washes. After the third wash, do not discard the supernatant yet, but keep it on the magnetic rack for later use.

7. Discard the supernatant of the Flag-tagged magnetic beads, add the remaining 40 μl protein in step 14, place on a rotating mixer, and incubate at room temperature for 2 hours.

8. After incubation, place the centrifuge tube on the magnetic rack. After the solution is clarified, discard the supernatant and wash the magnetic beads three times with 100 μL IP buffer.

9. Use 100 μL 3×FLAG peptide elution buffer to resuspend the Flag magnetic beads, place on a rotating mixer, and incubate at room temperature for 30 minutes.

10. After the incubation, place the centrifuge tube on the magnetic rack. After the solution is clarified, transfer the supernatant to a new centrifuge tube.

11. Discard the supernatant of Myc magnetic beads, use the Flag magnetic bead elution buffer in the previous step to resuspend the Myc magnetic beads, place on a rotating mixer, and incubate at room temperature for 2 hours.

12. After incubation, place the centrifuge tube on the magnetic rack. After the solution is clarified, discard the supernatant and wash the magnetic beads three times with 100 μL IP buffer.

13. After washing, use 100 μL IP buffer to resuspend the magnetic beads for later use to complete protein purification.

Among them, IP buffer 10ml:

10ml PBS (PH 7.2) + 50μl NP-40 (1/100 dilution) + 1 Roche protease inhibitor tablet

3×FLAG peptide elution buffer (400μg/ml working concentration) 250μl:

230μl IP Buffer + 20μl 3×FLAG peptide (concentration 5mg/ml).

16. Library affinity purification

Place the Myc magnetic beads + protein centrifuge tube purified in step 15 on the magnetic rack, and discard the supernatant after the solution is clarified.

Use the remaining DNA library that has passed the quality inspection in step 13 to resuspend the Myc magnetic beads, place them on a rotating mixer, and incubate at room temperature for 2 hours.

After incubation, place the centrifuge tube on a magnetic rack, wait for the solution to become clear, discard the supernatant, and wash the magnetic beads three times with 100 μL IP buffer.

After the last wash, discard the supernatant and resuspend the Myc magnetic beads in 30 μl 50 mM Tris-HCl.

Place the centrifuge tube in a metal bath and incubate at 98°C for 10 min to elute the bound DNA library.

After the incubation, the centrifuge tube was immediately placed on the magnetic rack. After the solution was clarified, the supernatant was transferred to a new centrifuge tube, which was the eluted affinity-purified DNA library.

17. Library Amplification after Affinity Purification

Configure the library amplification reagents according to the reaction system in Table 9:

Table 9

The nucleotide sequence of Universal Primer is 5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACAC GACGCTCTTCCGATC-3' (as shown in SEQ ID No. 11);

The nucleotide sequence of Index Primer is 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTT CAGACGTGTGCTCTTCCGATCT-3' (as shown in SEQ ID No. 12), wherein "XXXXXXXX" is an 8-base index sequence.

The amplification program is shown in Table 10:

Table 10

After PCR, add 45 μl Cleanup Beads 2.0, mix thoroughly, and let stand at room temperature for 5 minutes.

Place the centrifuge tube on a magnetic rack, wait for the solution to become clear, discard the supernatant, add 80% ethanol, and rinse twice.

Discard all ethanol and place the centrifuge tube on the magnetic rack to dry until the surface of the magnetic beads is matte.

Add 25 μl of enzyme-free water to resuspend the magnetic beads, mix thoroughly, and let stand at room temperature for 5 minutes.

Place the centrifuge tube on a magnetic rack, and after the solution is clarified, transfer the supernatant to a new centrifuge tube, which is the sequencing library after affinity purification.

18. Library Sequencing

The sequencing was performed using the Illumina platform Novasek 6000, with a sequencing strategy of PE150 and a sequencing volume of 6G.

The specific bioinformatics analysis method is as follows: NovaSeq was used to obtain raw data in PE150 bp fq format after high-throughput sequencing, and fastp (Shifu Chen et al., 2018) was used to filter the raw data to obtain high-quality sequencing data for downstream analysis.

The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical contents disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims (10)

1. The DNA affinity purification sequencing and library building method based on double Pull down is characterized by comprising the following steps of:

(1) Extracting genomic DNA of a target sample;

(2) DNA fragmentation is carried out on the genome DNA to obtain DNA fragments;

(3) Constructing a library of the DNA fragments to obtain a DNA library;

(4) Obtaining two transcription factor expression vectors with different labels;

(5) Placing two transcription factor expression vectors with equal quantity into the same in-vitro protein expression system, and carrying out in-vitro protein expression to obtain an expression product;

(6) Purifying the expression product sequentially by using different tag protein purification magnetic beads to obtain a purified product;

(7) Combining the purified product with the DNA library in the step (3), and sequentially incubating and eluting to obtain an eluted DNA library;

(8) Performing PCR amplification on the eluted DNA library to obtain a sequencing library;

(9) And (3) sequencing and analyzing the sequencing library.

2. The method for DNA affinity purification sequencing and library establishment based on double Pull down according to claim 1, wherein in the step (2), the size of the DNA fragment is 200-400 bp.

3. The method for constructing a library by double Pull down-based DNA affinity purification sequencing of claim 1, wherein in step (3), the library construction comprises end repair and linker ligation in sequence;

Preferably, the reaction system for end repair is: 0.8-1.2 mu L of DNA fragment, 2.8-3.2 mu L of NEXTflex ^TM End-pair & Adenylation Enzyme Mix 2.0,2.8-3.2 mu L of NEXTflex ^TM End-pair & Adenylation Buffer Mix.0, and NFW to 50 mu L;

Preferably, the reaction conditions for the end repair are: incubating at constant temperature of 18-22 ℃, 28-32 min-64-66 ℃, 28-32 min-3-5 ℃;

preferably, the reaction system for the joint connection is: 48-52 mu L of end repair product, 2.8-3.2 mu L of genobaits joint, 44-45 mu L of NEXTflex ^TM Ligase Enzyme Mix and 2.8-3.2 mu L of NEXTflex ^TM Ligase Buffer Mix;

preferably, the reaction conditions for the linker ligation are: incubating at constant temperature of 18-22 deg.c for 14-16 min to 3-5 deg.c.

4. The method for DNA affinity purification sequencing and pooling based on double Pull down of claim 1, wherein step (4) comprises:

s1, selecting a transcription factor 1 and a transcription factor 2;

S2, respectively designing PCR amplification primers according to coding CDS sequences of the transcription factor 1 and the transcription factor 2;

S3, respectively amplifying the coding CDS sequence of the transcription factor 1 and the coding CDS sequence of the transcription factor 2 by using PCR amplification primers;

S4, respectively constructing a coding CDS sequence of the transcription factor 1 and a coding CDS sequence of the transcription factor 2 on the protein expression vector 1 and the protein expression vector 2;

S5, respectively converting the protein expression vector 1 and the protein expression vector 2 into escherichia coli for culture;

S6, selecting monoclonal colonies for sequencing, and screening out expression vectors consistent with coding CDS sequences of the transcription factors 1 and 2 to obtain expression vector strains;

S7, extracting a protein expression plasmid of the transcription factor 1 and a protein expression plasmid of the transcription factor 2 to obtain two transcription factor expression vectors with different labels;

in the step S1, the interaction relationship exists between the transcription factor 1 and the transcription factor 2, so that the expression of the same gene can be regulated and controlled together.

5. The DNA affinity purification sequencing library building method based on double Pull down according to claim 4, wherein in the step S2, the coding CDS sequence of the transcription factor 1 is shown as SEQ ID No. 1;

Preferably, the coding CDS sequence of transcription factor 2 is shown as SEQ ID No. 2;

Preferably, in step S3, the amplification primers of the CDS sequence encoding the transcription factor 1 are shown as SEQ ID No.3 and SEQ ID No. 4;

preferably, the amplification primers of the CDS sequence encoding transcription factor 2 are shown in SEQ ID No.5 and SEQ ID No. 6.

6. The method for DNA affinity purification sequencing and banking based on double Pull Down according to claim 4, wherein in step S4, the initial expression vectors of the protein expression vector 1 and the protein expression vector 2 are independently pf3k;

preferably, the protein expression vector 1 is Flag tagged;

the upstream sequence of the Flag tag is shown as SEQ ID No. 7;

The downstream sequence of the Flag tag is shown as SEQ ID No. 8;

preferably, the protein expression vector 2 carries a Myc tag;

the upstream sequence of the Myc tag is shown as SEQ ID No. 9;

The downstream sequence of the Myc tag is shown as SEQ ID No. 10.

7. The method for DNA affinity purification sequencing and pooling based on double Pull down according to claim 1, wherein in step (5), the reagent for in vitro protein expression comprises: 28-32 mu L of TNT mix, 0.8-1.2 mu L of each of two transcription factor expression vectors, and no enzyme water to 50 mu L;

preferably, the temperature of in vitro protein expression is 28-32 ℃;

the time for in vitro protein expression is 1.8-2.2 h.

8. The method for sequencing and library establishment based on double Pull down DNA affinity purification of claim 1, wherein in step (6), the different tag protein purification magnetic beads comprise Flag tag purification magnetic beads and Myc tag purification magnetic beads;

Preferably, in the step (7), the incubation time is 1.8-2.2 h.

9. The method for DNA affinity purification sequencing and banking based on double Pull down according to claim 1, wherein in step (8), the reaction system of PCR amplification comprises: 14 to 16 mu L of eluted DNA library, 0.8 to 1.2 mu L of Universal Primer,0.8 to 1.2 mu L of Index Primer,24 to 26 mu L of 2 xKAPA HIFI mix, and no enzyme water to 50 mu L;

preferably, the nucleotide sequence of the Universal Primer is shown as SEQ ID No. 11;

the nucleotide sequence of the Index Primer is shown as SEQ ID No. 12;

Preferably, in step (8), the reaction procedure of the PCR amplification is ：97～99℃,2.5～3.5min,1cycle→97～99℃,9～11s,58～62℃,28～32s,70～73℃,28～32s,28～32cycle→70～73℃,1.5～2.5min,1cycle→3～5℃, for use at constant temperature.

10. The method for DNA affinity purification sequencing and banking based on double Pull down according to claim 1, wherein in the step (9), the sequencing strategy is PE150, and the sequencing amount is 5.8-6.2G.