A DAP-seq experimental method for constructing a fusion transposase library

Abstract

The present application discloses a DAP-seq experimental method for building a library with a fusion transposase, which uses Tn5 transposase to bind to a DNA fragment region that is not protected by transcription factor (TF) binding, produces an enzyme cutting effect, and completes the library construction, cooperates with the corresponding bioinformatics analysis method, and locks the transcription factor binding site (TFBS) and the binding motif (motif). The DAP-seq experimental method for building a library with a fusion transposase described in the present application utilizes the property that the transcription factor binding site is not protected by the transcription factor binding, uses Tn5 transposase to cut the DNA fragment region that is not protected by the transcription factor binding and completes the library construction, cooperates with the corresponding bioinformatics analysis method, and realizes the precise positioning of TFBS and the direct locking of highly reliable motif sequences.

Description

Technical Field

[0001] The invention relates to the technical field of protein precipitation and enrichment, and in particular to a DAP-seq experimental method for building a library of a fusion transposase. Background Art

[0002] Transcription factors (TFs) are a hot topic in the study of gene expression regulation. Comprehensive identification of transcription factor binding sites (TFBSs) in the genome and summarizing the characteristics of TF binding motifs are crucial for analyzing the functions of TFs. Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful method for discovering TFBSs. ChIP-seq experiments rely on high-quality antibodies. It is difficult to prepare antibodies for rare or low-expressed proteins, and non-model organisms lack available commercial antibodies. Therefore, CHIP-seq is only suitable for large-scale development in common TFs in model organisms.

[0003] DNA affinity purification sequencing (DAP-seq) is a technology that can be carried out on a large scale and partially replace CHIP-seq. DAP-seq completes the enrichment and sequencing of TF-bound DNA fragments by combining genomic DNA fragments with modified TF proteins in vitro, thereby achieving the detection of TFBS. DAP-seq is not restricted by antibodies and species, and has the advantage of high throughput. Since its inception, this technology has been widely used in the study of transcriptional regulation and epigenomics.

[0004] However, DAP-seq still faces difficulties in locking TFBS and motifs in practical applications. The DAP-seq method is usually used in non-model organisms, and the motif features of the target TF may not be exactly the same as those of the model organisms. The length of the DNA fragment used for DAP-seq enrichment is usually 300bp to 500bp, while the actual length of the TFBS is usually only 10bp. Therefore, DAP-seq can only perform peak analysis through bioinformatics. The width of the peak is usually 300bp to 500bp, which can only provide an approximate range of the target TFTFBS, but cannot accurately locate the TFBS. The overly wide peak range makes the motif enrichment analysis based on the peak area full of noise. The results of the peak-based motif enrichment analysis are usually full of noise, and can only provide a list of a series of candidate TF motifs, but cannot directly lock a highly reliable motif sequence.

[0005] Therefore, the conventional DAP-seq experimental method needs to be further improved to solve the problem of insufficient accuracy of TFBS detection and inability to directly determine the transcriptome factor motif. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a DAP-seq experimental method for building a library with a fusion transposase, so as to solve the problem of insufficient TFBS detection accuracy and inability to directly determine the transcriptome factor motif.

[0007] To solve the above problems, the technical solutions adopted in this application are as follows:

[0008] The embodiment of the present application provides a DAP-seq experimental method for building a library by fusion transposase, which uses Tn5 transposase to bind to DNA fragment regions that are not protected by transcription factor binding to produce an enzyme cutting effect and complete library construction, and cooperates with corresponding bioinformatics methods to lock transcription factor binding sites and binding motifs.

[0009] As a further preferred solution, the DAP-seq experimental method for constructing a fusion transposase library described in the examples of the present application includes:

[0010] Expression and enrichment of transcription factor proteins: Wheat germ reaction system was used for protein expression. After expression, magnetic bead solution containing halo-tag antibody was used for protein enrichment to obtain transcription factor proteins with halo-tag label.

[0011] DNA fragmentation and fragment screening: After the DNA sample is fragmented, the DNA fragments are screened using a magnetic bead solution containing halo-tag antibodies;

[0012] Protein affinity adsorption of DNA fragments: adding the above-mentioned transcription factor protein-adsorbed DNA fragments to the above-screened DNA fragments to obtain transcription factor-bound DNA;

[0013] Treating TF-bound DNA with Tn5 transposase: Treating the DNA bound by the above transcription factors with Tn5 transposase, and Tn5 transposase binds to the DNA fragment region that is not protected by the transcription factor binding;

[0014] Construction of DNA library: Using PCR reaction system, construct DNA library through PCR amplification;

[0015] Sequencing and bioinformatics analysis: The products treated with Tn5 transposase were sequenced and analyzed using software to screen out transcription factor binding motifs.

[0016] As a further preferred solution, the transcription factor protein expression and enrichment described in the embodiments of the present application includes the following steps:

[0017] 1) Using wheat germ reaction system to conduct metal bath;

[0018] 2) After the wheat germ protein expression system reaction is completed, it is placed at room temperature, and the equilibration buffer is added thereto to obtain mixed solution I; at the same time, the EP tube of the equilibration buffer added with halo-tag magnetic beads is placed on a magnetic stand, the supernatant is discarded, and the mixed solution I is added, and the mixture is shaken and mixed at room temperature and incubated;

[0019] 3) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the adhered magnetic beads are washed off and settled to the bottom, and discard the supernatant; immediately add the equilibration buffer, shake with a mixer, take out and place on a magnetic rack to stand, and discard the supernatant;

[0020] 4) Repeat the above steps 1)-3) to obtain a transcription factor protein with a halo-tag label.

[0021] As a further preferred scheme, the transcription factor protein expression and enrichment described in the embodiments of the present application also includes preparing magnetic beads with halo-tag antibodies during the reaction of the wheat germ protein expression system: according to the amount of 10 μL of magnetic beads required for each protein sample, the corresponding volume of magnetic beads is taken out according to the number of samples, and the beads are washed on a magnetic rack with equilibrium buffer in an EP tube, and finally PBS solution is added and evenly distributed in a corresponding number of EP tubes.

[0022] As a further preferred solution, the DNA fragmentation and fragment screening described in the embodiments of the present application include the following steps:

[0023] 1) Shear the DNA sample, transfer the sheared DNA into an EP tube, add the equilibrium buffer solution containing magnetic beads, mix well, and place at room temperature;

[0024] 2) When the solution is clear, transfer the supernatant to a new EP tube. During this process, the original centrifuge tube does not leave the magnetic rack;

[0025] 3) Add the equilibration buffer solution containing magnetic beads to the supernatant, pipette and mix well, and place on a magnetic stand at room temperature;

[0026] 4) When the solution is clear, discard the supernatant, that is, add solvent to wash, let it stand for stratification and then discard the solvent; absorb the remaining liquid and leave it at room temperature;

[0027] 5) After the magnetic beads are dried, add nuclease-free water, remove the EP tube from the magnetic stand, pipette and mix, and dissolve at room temperature;

[0028] 6) After dissolution, place the EP tube on a magnetic rack and let it stand. After stratification, transfer the supernatant to a new EP tube;

[0029] 7) Quantum bit quantification, measuring the concentration of recovered DNA, and obtaining fragmented DNA;

[0030] As a further preferred solution, during the DNA fragmentation and fragment screening described in the embodiments of the present application, DNA fragmentation is performed using the instrument Bioruptor Plus, and the fragmentation conditions are: temperature 4°C, on 30s / off 30s as one cycle, and the length of the fragmented DNA is 200bp-600bp.

[0031] As a further preferred solution, the obtaining of protein affinity-adsorbed DNA fragments described in the embodiments of the present application comprises the following steps:

[0032] 1) Take the equilibrium buffer solution into a dry EP tube, add the above-mentioned fragmented DNA into it, and mix by pipetting to form mixed solution II;

[0033] 2) Discard the equilibrium buffer solution in the magnetic beads on the magnetic stand, add the above-mentioned transcription factor protein with halo-tag label, and incubate on a mixer;

[0034] 3) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the equilibration buffer, shake on a mixer, take out and place on a magnetic rack, and discard the supernatant;

[0035] 4) Repeat the above steps 1) to 3) to obtain DNA bound to the transcription factor.

[0036] As a further preferred solution, in the embodiment of the present application, the treatment of TF-bound DNA based on Tn5 transposase includes the following steps:

[0037] 1) The residual solution in the DNA fragments adsorbed by the above protein affinity was sucked out, and Tn5 transposase reaction solution was directly added for incubation;

[0038] 2) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the equilibration buffer solution, shake on a mixer, take out and place on a magnetic rack until the layers are separated, and discard the supernatant;

[0039] 3) Repeat the above steps 1)-2) to obtain the product after Tn5 transposase treatment.

[0040] As a further preferred solution, the construction of the DNA library described in the embodiment of the present application comprises the following steps:

[0041] 1) Aspirate the residual solution and add the PCR reaction system directly;

[0042] 2) Measure the PCR product into a new centrifuge tube, add 1.05× magnetic beads into the tube, pipette and mix, and place on the BLOCK plate at room temperature for 5-15 minutes;

[0043] 3) Then place the centrifuge tube on a magnetic rack to separate layers, discard the supernatant, immediately add ethanol to wash, and after separation; discard the supernatant, immediately add ethanol, discard the supernatant, and suck out the remaining ethanol;

[0044] 4) Place on the magnetic rack and add dd H after the magnetic beads are dry. 2 O, remove the magnetic stand and pipette repeatedly;

[0045] 5) After standing, place on a magnetic rack to stand and stratify, transfer the supernatant to a new EP tube and freeze it for storage.

[0046] As a further preferred solution, the sequencing and bioinformatics analysis described in the embodiments of the present application include

[0047] Data comparison: Use alignment software to align the data to the reference genome for comparison, filter out reads with low alignment quality values, use the unique alignment sequence obtained after alignment as the analysis object, analyze its coverage distribution on the reference genome, count the depth information of the genomic sites, and obtain the statistical results of the sequencing depth on the genome;

[0048] Information analysis: After sequencing, the DNA product is used to find the binding sites between the target protein and DNA in the whole genome. The analysis software is used to perform peak scanning in the whole genome, and the peak information on the genome is analyzed to screen out peak-related genes.

[0049] Motif enrichment analysis was performed based on transcription factors: within each peak, the number of Tn5 transposase insertion sites in the window was counted with a window size of 20bp and a sliding step size of 5bp, and adjacent windows were used as units; within each peak, 50bp at both ends of the peak were excluded, and the window with the lowest insertion fragment depth was found as the candidate window containing the transcription factor binding site; for the top 500 peaks ranked by sequencing depth, candidate windows containing transcription factor binding sites were extracted, and motif enrichment analysis was performed to screen protein-binding motifs.

[0050] Compared with the prior art, the beneficial effect of the present invention is that the DAP-seq experimental analysis method for building a library with a fusion transposase described in the present application utilizes the property that the transcription factor binding site (TFBS) is not protected by the binding of the transcription factor (TF), uses Tn5 transposase to cut the DNA fragment region that is not protected by the binding of the transcription factor (TF) and completes the library construction, and cooperates with the corresponding bioinformatics analysis method to achieve the precise positioning of the TFBS and directly lock the highly reliable motif sequence.

[0051] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0053] Figure 1 The results of motif enrichment analysis based on transcription factor binding site (TFBS) related windows.

[0054] Figure 2 The peak structure of the peak detected by the experimental sequencing results in Example 1 (the red arrow is the potential TFBS); the sample group is the upper 3 sub-graphs, and the trough area pointed by the arrow is the expected TFBS; the control group is the lower 3 sub-graphs.

[0055] Figure 3 This is a motif map of the top 10 enrichment levels of sample 1 in Application Example 1.

[0056] Figure 4 This is a motif map of the top 10 enrichment levels of sample 2 in Application Example 1.

[0057] Figure 5 The top 10 motifs with the highest enrichment in the sample group control in Example 1 were used. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0059] The term "comprising" and other equivalent descriptions involved in the specification and claims of this application are intended to cover non-exclusive inclusions, including both the contents clearly described in the specification and claims and the steps that are not described in the specification and claims but are inherent in the product, method or structure.

[0060] The present application embodiment provides a DAP-seq experimental method for building a library by fusion transposase, using Tn5 transposase to bind to DNA fragment regions that are not protected by transcription factor binding, produce enzyme cutting effects, and complete library construction, cooperate with corresponding bioinformatics analysis methods to lock transcription factor binding sites and binding motifs. In the Tn5 transposase, only the OE sequence can be recognized and randomly inserted into DNA to complete in vitro transposition. In the present application embodiment, when Tn5 is used for sequencing library construction, multi-step reactions such as DNA fragmentation, end repair, and connector connection can be converted into a one-step reaction, greatly shortening the library construction time, improving work efficiency, and shortening the library construction time.

[0061] Specifically, the DAP-seq experimental method for constructing a fusion transposase library described in the examples of the present application includes:

[0062]

Transcription factor protein expression and enrichment

[0063] 1) Using wheat germ reaction system, metal bath is performed. In this step, the reaction conditions are: reaction temperature is 25℃-30℃, reaction time is 1.5-3h. In this step, HALO-GFP vector can also be selected as a positive control to detect whether the wheat germ expression system works;

[0064] 2) During the wheat germ protein expression system, start preparing magnetic beads containing halo-tag antibodies;

[0065] 3) After the wheat germ protein expression system reaction is completed, it is placed at room temperature, and an equilibrium buffer (PBS and 10 mM imidazole, pH 7.4) is added thereto to provide a suitable pH and ionic strength to promote the binding of the target molecule and the ligand, thereby obtaining a mixed solution I; at the same time, an EP tube containing the equilibrium buffer solution with halo-tag magnetic beads is placed on a magnetic stand, the supernatant is discarded, and the mixed solution I is added, and the mixture is shaken and mixed at room temperature and incubated;

[0066] 4) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the adhered magnetic beads are washed off and settled to the bottom, and discard the supernatant; immediately add the equilibration buffer, shake with a mixer, take out and place on a magnetic rack to stand, and discard the supernatant;

[0067] 5) Repeat the above steps 1)-3) to obtain a transcription factor protein with a halo-tag label.

[0068] In some embodiments of the present application, the wheat germ reaction system comprises wheat germ system buffer and HALO-tag plasmid. Preferably, the present application embodiment adopts 25 μL wheat germ reaction system, including 15 μL wheat germ system buffer and 10 μL HALO-tag plasmid.

[0069] The HaloTag tag protein used in the embodiments of the present application is a genetically modified derivative of a dehalogenase, which can be effectively covalently bound to a variety of synthetic HaloTag ligands. The molecular weight of the HaloTag tag protein is 33KDa, which can be fused to the N-terminus or C-terminus of the recombinant protein and expressed in prokaryotic and eukaryotic systems. The HaloTag ligand is a small molecule chemical that can covalently bind to the HaloTag protein in vitro or in vivo. These ligands are composed of two key components: a universal HaloTag reaction linker that binds the HaloTag protein; a functional group, such as a fluorescent dye or an affinity medium. The ability to covalently and specifically bind to a variety of synthetic reporter groups and affinity ligands enables the HaloTag protein to be used for detection and affinity binding or solid-phase fixation of target proteins.

[0070]

DNA fragmentation and fragment screening

[0071] 1) Take the DNA sample and put it in an EP thin-walled tube. Use the instrument Bioruptor Plus to shear the DNA. The shearing conditions are: temperature 4°C, on 30s / off 30s as one cycle, 8-15 cycles, the sheared DNA fragment length is 200bp-600bp. Transfer the sheared DNA to an EP tube, add the equilibrium buffer solution containing magnetic beads, mix well, and place at room temperature;

[0072] 2) Place the sample on the magnetic stand and let it stand. When the solution is clear, transfer the supernatant to a new EP tube. During this process, the original centrifuge tube does not leave the magnetic stand.

[0073] 3) Add the equilibration buffer solution containing magnetic beads to the supernatant, pipette and mix well, and place on a magnetic stand at room temperature;

[0074] 4) When the solution is clear, discard the supernatant, that is, add a solvent for washing, let it stand for stratification and then discard the solvent; absorb the remaining liquid and leave it at room temperature; in this step, the solvent is 80% ethanol;

[0075] 5) After the magnetic beads are dried, add nuclease-free water, remove the EP tube from the magnetic stand, pipette and mix, and dissolve at room temperature;

[0076] 6) After dissolution, place the EP tube on a magnetic rack and let it stand. After stratification, transfer the supernatant to a new EP tube;

[0077] 7) Quantum bit quantification, measuring the concentration of recovered DNA, and obtaining fragmented DNA.

[0078] [Obtaining protein affinity-adsorbed DNA fragments]

[0079] 1) Take the equilibrium buffer solution into a dry EP tube, add the above-mentioned fragmented DNA into it, and mix by pipetting to form mixed solution II;

[0080] 2) Discard the equilibrium buffer solution in the magnetic beads on the magnetic stand, add the above-mentioned transcription factor protein with halo-tag label, and incubate on a mixer;

[0081] 3) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the equilibration buffer, shake on a mixer, take out and place on a magnetic rack, and discard the supernatant;

[0082] 4) Repeat the above steps 1) to 3) 2 to 5 times to obtain DNA bound to the transcription factor.

[0083]

Tn5 transposase processes TF-bound DNA

[0084] 1) The residual solution in the DNA fragments adsorbed by the above protein affinity was sucked clean, and Tn5 transposase reaction solution was directly added for incubation; the incubation temperature was about 37°C;

[0085] 2) After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the equilibration buffer solution, shake on a mixer, take out and place on a magnetic rack until the layers are separated, and discard the supernatant;

[0086] 3) Repeat the above steps 1)-2) to obtain the product after Tn5 transposase treatment.

[0087]

Construction of DNA library

[0088] 1) Aspirate the residual solution and add the PCR reaction system directly;

[0089] 2) Measure the PCR product into a new centrifuge tube, add 1.05× magnetic beads into the tube, pipette and mix, and place on the BLOCK plate at room temperature for 5-15 minutes;

[0090] 3) Then place the centrifuge tube on a magnetic rack to separate layers, discard the supernatant, immediately add ethanol to wash, and after separation; discard the supernatant, immediately add ethanol, discard the supernatant, and suck out the remaining ethanol;

[0091] 4) Place on the magnetic rack and add dd H after the magnetic beads are dry. 2 O, remove the magnetic stand and pipette repeatedly;

[0092] 5) After standing, place on a magnetic rack to stand and stratify, transfer the supernatant to a new EP tube and freeze it for storage.

[0093] In the present application example, the PCR reaction system is as follows:

[0094]

[0095] The reaction procedure is as follows:

[0096]

[0097] Place on ice.

[0098]

Sequencing and bioinformatics analysis

[0099] Data comparison: Use alignment software to align the data to the reference genome for comparison, filter out reads with low alignment quality values, use the unique alignment sequence obtained after alignment as the analysis object, analyze its coverage distribution on the reference genome, count the depth information of the genomic sites, and obtain the statistical results of the sequencing depth on the genome;

[0100] Information analysis: After sequencing, the DNA product is used to find the binding sites between the target protein and DNA in the whole genome. The analysis software is used to perform peak scanning in the whole genome, and the peak information on the genome is analyzed to screen out peak-related genes.

[0101] Motif enrichment analysis was performed based on transcription factors: within each peak, the number of Tn5 transposase insertion sites in the window was counted with a window size of 20bp and a sliding step size of 5bp, and adjacent windows were used as units; within each peak, 50bp at both ends of the peak were excluded, and the window with the lowest insertion fragment depth was found as the candidate window containing the transcription factor binding site; for the top 500 peaks ranked by sequencing depth, candidate windows containing transcription factor binding sites were extracted, and motif enrichment analysis was performed to screen protein-binding motifs.

[0102] The following are specific embodiments of the present application. In the following embodiments, the reagents, equipment, software, etc. used can be obtained through market sales channels unless otherwise specified in the present application.

[0103] Example 1

[0104] This example provides a DAP-seq experimental method for building a library using a fusion transposase, which uses Tn5 transposase to bind to DNA fragment regions that are not protected by transcription factors to produce an enzyme cutting effect and complete library construction, and cooperates with the corresponding bioinformatics analysis method to lock the transcription factor binding sites and binding motifs. The specific steps are as follows:

[0105] 1. Expression and enrichment of transcription factor proteins

[0106] 1) Use 25 μL wheat germ reaction system, which contains 15 μL wheat germ system buffer and 10 μL HALO-tag plasmid, and perform metal bath at 25°C for 2 hours. In this step, HALO-GFP vector can be selected as a positive control to detect whether the wheat germ expression system works.

[0107] 2) At 1.5h of protein expression, start preparing magnetic beads containing halo-tag antibodies. Each protein sample requires 10μl of magnetic beads. Take out the corresponding volume of magnetic beads according to the number of samples, wash them 4 times at room temperature with Equilibration buffer in a 1.5ml EP tube, and operate on a magnetic stand. Finally, add PBS solution and evenly distribute it in the corresponding number of EP tubes, and remove the supernatant before use.

[0108] 3) After the wheat germ protein expression system has finished reacting, place it at room temperature and add 25 μL Equilibration buffer. Meanwhile, place the EP tube containing the beads Equilibration buffer solution on a magnetic rack for 2-5 minutes, discard the supernatant, add the above 50 μL mixed solution (mixed solution: 25 μL wheat germ expression protein and 25 μL Equilibration buffer), and shake it at room temperature at 25°C and 1500 rpm in a mixer for 1 hour.

[0109] 4) After incubation, place the tube on a magnetic rack at room temperature, use a pipette to absorb the supernatant to rinse the tube wall so that all the adhered beads are washed off and settled to the bottom, discard the supernatant, immediately add 50 μL of Equilibration buffer, shake at 25℃ and 1500 rpm for 1 min, take out and place on a magnetic rack for 30 sec, and discard the supernatant.

[0110] 5) Repeat the above steps twice, washing 3 times in total.

[0111] 2. DNA fragmentation and fragment screening

[0112] 1) 100 μL DNA sample was placed in a 0.5 ml EP thin-walled tube and sheared using the Bioruptor Plus instrument at 4°C, one cycle of on 30s / off 30s, 10 cycles, and 1.5% agarose gel electrophoresis to detect the shearing status. The sheared DNA fragment length was controlled at 200bp-600bp.

[0113] 2) Transfer the sheared DNA sample to a 1.5 ml EP tube, adjust the volume to 100 μl, add 60 μl of magnetic bead solution (shake well at room temperature), pipette 10 times to mix, and leave at room temperature for 5-15 minutes.

[0114] 3) Place the sample on the magnetic rack and leave it at room temperature for 5 minutes. When the solution is clear, transfer the supernatant to a new 1.5 ml EP tube. During this process, the original centrifuge tube does not leave the magnetic rack.

[0115] 4) Add 60 μL of magnetic bead solution to the supernatant, pipette 10 times to mix, and leave at room temperature for 5-15 minutes.

[0116] 5) Place the sample on a magnetic rack and leave it at room temperature for 5 minutes. When the solution is clear, discard the supernatant with a pipette, immediately add 200 μL 80% ethanol, leave it for a few seconds, discard the 80% ethanol with a pipette, add 200 μL 80% ethanol again to wash it, discard the 80% ethanol with a pipette, and then use a 10 μL gun to absorb the remaining liquid and leave it at room temperature for 5 minutes.

[0117] 6) After the magnetic beads are dried, add 30-50 μL of nuclease free water, remove the EP tube from the magnetic stand, pipette and mix, and dissolve at room temperature for 5 minutes.

[0118] 7) Place the sample on a magnetic rack for 5 min and transfer the supernatant to a new 1.5 mL EP tube.

[0119] 8) Quantum bit quantification and measurement of recovered DNA concentration.

[0120] 3. Protein affinity adsorption of DNA fragments

[0121] 1) Take 25 μL of Equilibration buffer and add it to a clean 1.5 mL EP tube. Then add 25 μL of fragmented DNA and mix by pipetting.

[0122] 2) At this time, discard the Equilibration buffer in the magnetic beads on the magnetic rack, add the mixed solution prepared in step 5, and shake at 1500 rpm at 25℃ for 1 hour.

[0123] 3) After incubation, place the tube on a magnetic rack at room temperature, use a pipette to aspirate the supernatant to rinse the tube wall so that all beads sink to the bottom, discard the supernatant, immediately add 50 μL of Equilibration buffer, shake at 1500 rpm at 25°C for 1 min, take out and place on a magnetic rack for 30 sec, and discard the supernatant.

[0124] 4) Repeat the above steps twice, a total of 3 times.

[0125] 4. Tn5 transposase processes TF-bound DNA

[0126] 1) Use a 10 μL pipette to suck out the remaining solution. Add 30 μL Tn5 enzyme reaction solution directly without drying and incubate at 37°C for 30 min.

[0127] 2) After incubation, place the tube on a magnetic rack at room temperature, use a pipette to aspirate the supernatant to rinse the tube wall so that all beads sink to the bottom, discard the supernatant, immediately add 50 μL of Equilibration buffer, shake at 1500 rpm at 25°C for 1 min, take out and place on a magnetic rack for 30 sec, and discard the supernatant.

[0128] 3) Repeat the above steps twice, for a total of 3 times.

[0129] 5. Construction of DNA Library

[0130] 1) Use a 10μL pipette to suck out the remaining solution and add 40μL of PCR reaction system directly without drying.

[0131] 2) Use a 10 μL pipette to quantify the PCR product in the PCR tube into a new 1.5 mL centrifuge tube, add 1.05× magnetic beads (for example, 25 μL PCR product plus 26.3 μL magnetic beads), pipette to mix, and place on a BLOCK plate at room temperature for 5-15 minutes.

[0132] 3) Place on the magnetic rack for 5 min, discard the supernatant, and immediately add 200 μL 80% ethanol.

[0133] 4) Discard the supernatant, immediately add 200 μL 80% ethanol, discard the supernatant, and use a 10 μL pipette to remove the remaining ethanol.

[0134] 5) Place on the magnetic rack for 5 minutes. After the beads are dry, add 20 μL dd H 2 O, remove the magnetic stand and pipette up and down 10 times.

[0135] 6) Let stand for 5 min, place on a magnetic rack for 5 min, transfer the supernatant to a new 1.5 mL EP tube, and store at -20°C.

[0136] 6. Sequencing and bioinformatics analysis

[0137] 1) Data alignment: The alignment software Bowtie 2 (version: 2.2.5) was used to align the data to the reference genome, filter out reads with low alignment quality values, and finally use the reads aligned to the only position on the genome (unique alignment reads) for subsequent information analysis. The unique alignment sequence obtained after alignment was used as the analysis object, and its coverage distribution on the reference genome was analyzed, and the depth information of the genomic sites was counted to obtain the statistical results of the sequencing depth on the genome.

[0138] 2) Peak analysis: Sequencing and analyzing the DNA products after DAP, searching for the binding sites of the target protein and DNA in the whole genome, using MACS2 (version: 2.1.2) analysis software to perform peak calling (DAPSequencing enriched regions) in the whole genome, with a threshold of q-value < 0.05, and analyzing the position information of the peak on the genome, the sequence information of the peak region, etc., to screen out the peak-related genes.

[0139] 3) Conduct motif enrichment analysis based on TFBS: In each peak, the window size is 20bp, the sliding step is 5bp, and the adjacent window is used as the unit to count the number of Tn5 enzyme insertion sites in the window. In each peak, the 50bp at both ends of the peak are excluded, and the window with the lowest insertion fragment depth is found as the candidate window containing TFBS. For the top 500 peaks in sequencing depth, candidate windows containing TFBS are extracted and motif enrichment analysis is performed to screen protein-binding motifs. Results can be found in Figure 1 .

[0140] Application Example 1

[0141] The gene used in the application example is soybean transcription factor ER3 (GLYMA.14G050100). The motif of the transcription factor is known (CGCCGCC), so it can be used to judge the accuracy of the experimental results.

[0142] The experimental design is as follows:

[0143] New method group: The DAP-seq experimental method described in Example 1 of the present application was used to conduct the experiment, and two experimental replicates were set up, which were named sample groups 1 and sample 2 respectively.

[0144] Control group: The experiment was conducted using the traditional DAP-seq experimental method and was named the control group.

[0145] Experimental methods:

[0146] The DAP-seq experimental method described in Example 1 of the present application is based on the Tn5 enzyme to complete the cutting of genomic DNA fragments, enrichment of target DNA fragments and library construction. The control group uses the traditional DAP-seq method, which uses ultrasonic shearing of genomic DNA before enrichment and library construction of target DNA. The specific operations of enrichment and library construction follow the same steps as in Example 1.

[0147] Sequencing and analysis content:

[0148] After completing the construction of the sequencing library and sequencing, 6G data was detected for each sample, and peak detection, peak type analysis, and motif enrichment analysis were performed.

[0149] Implementation results:

[0150] (1) Peak shape comparison

[0151] The peaks obtained by analysis are visualized. The sequencing depth distribution of the Peak region shows an obvious bimodal structure (such as Figure 3 ), the valley in the middle of the double peak is the TFBS. In the peak shape of the sample group, the valley in the middle of the double peak is the area corresponding to the TFBS (transcription factor binding site). The DNA in this area is protected by TF binding, and the Tn5 transposase cannot directly cut this site, so the sequencing depth is lower than the surrounding DNA open area. In the control group, no similar double peak structure was detected. Based on the 20bp windows of the peak and valley area, motif enrichment analysis can directly obtain a motif with extremely significant specificity, which is consistent with the ERF transcription factor motif reported in the literature.

[0152] (2) Motif enrichment analysis

[0153] The binding motif of the transcription factor can be obtained through motif enrichment analysis. For the new method group, the motif enrichment analysis was performed based on 20bp windows in the peak and valley regions. In the control group, since there was no peak and valley structure, the motif enrichment analysis was performed using the entire peak region.

[0154] Among the top ten motifs with the highest enrichment in the two samples, eight are motifs of ERF family transcription factors that have been reported ( Figure 3 , Figure 4 ), and the motif enrichment was highly significant, with a P value < 1×10 -100 The top three motifs are all the classic motif sequences of the ERF family, CGCCGCC. Even if the true motif of the transcription factor is unknown, we can use the top three motifs to correctly determine the motif of the transcription factor.

[0155] In the control group, only one ERF family motif was among the top 10 motifs in terms of enrichment, ranking low at only ninth place, and the enrichment was weakly significant (P value = 3.15 × 10 -4 ). The first motif is CCACCG, which is quite different from the real motif of the transcription factor. We cannot correctly determine the motif of the transcription factor using the top three motifs.

[0156] Based on the above results, we can conclude that the experimental method described in this application improves the specificity of TFBS enrichment, reduces background noise, and is conducive to accurately determining the motif of the target transcription factor in the experimental results.

[0157] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A DAP-seq experimental method for building a library of fusion transposases, characterized in that: The Tn5 transposase is used to bind to the DNA fragment region that is not protected by the transcription factor binding, produce an enzyme cutting effect, and complete the library construction, and cooperate with the corresponding bioinformatics analysis method to lock the transcription factor binding site and binding motif, including the following steps: Expression and enrichment of transcription factor proteins: The wheat germ reaction system was used for protein expression. After the expression was completed, the magnetic bead solution containing the halo-tag antibody was used for protein enrichment to obtain transcription factor proteins with halo-tag labels. The expression and enrichment of transcription factor proteins included the following steps: S11 uses a wheat germ reaction system followed by a metal bath; S12 After the wheat germ protein expression system has finished reacting, it is placed at room temperature, and the equilibration buffer is added thereto to obtain a mixed solution I; at the same time, the EP tube containing the equilibration buffer solution with halo-tag magnetic beads is placed on a magnetic stand, the supernatant is discarded, and the mixed solution I is added, and the mixture is shaken and mixed at room temperature and incubated; S13 After the incubation is completed, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the attached magnetic beads are washed off and settled to the bottom, and discard the supernatant; immediately add the equilibration buffer, shake with a mixer, take out and place on a magnetic rack to stand, and discard the supernatant; S14 repeats the above steps S11-S13 to obtain a transcription factor protein with a halo-tag label; DNA fragmentation and fragment screening: After the DNA sample is broken, the DNA fragments are screened using a magnetic bead solution containing halo-tag antibodies; DNA fragmentation and fragment screening includes the following steps: S21 shears the DNA sample, transfers the sheared DNA to an EP tube, adds a balanced buffer solution containing magnetic beads, mixes well, and leaves at room temperature. The DNA shearing is performed using the instrument BioruptorPlus. The shearing conditions are: temperature 4°C, on 30s / off 30s as one cycle, and the sheared DNA fragment length is 200bp-600bp; S22 When the solution is clear, transfer the supernatant to a new EP tube. During this process, the original centrifuge tube does not leave the magnetic rack. S23 Add the equilibration buffer solution containing magnetic beads to the supernatant, pipette and mix, and place on a magnetic stand at room temperature; S24 When the solution is clear, discard the supernatant, that is, add a solvent for washing, let it stand for stratification and then discard the solvent; absorb the remaining liquid and leave it at room temperature; S25 After the magnetic beads are dried, add nuclease-free water, remove the EP tube from the magnetic stand, pipette and mix, and dissolve at room temperature; After S26 is dissolved, place the EP tube on a magnetic rack and let it stand. After stratification, transfer the supernatant to a new EP tube. S27 quantum bit quantification, measuring the concentration of recovered DNA, and obtaining fragmented DNA; Protein affinity adsorption of DNA fragments: adding the above-mentioned transcription factor protein adsorption DNA fragments to the screened DNA fragments to obtain transcription factor-bound DNA; protein affinity adsorption of DNA fragments includes the following steps: S31 Take the equilibrium buffer solution into a dry EP tube, add the above-mentioned fragmented DNA into it, and mix by pipetting to form a mixed solution II; S32 discard the equilibrium buffer solution in the magnetic beads on the magnetic stand, add the above-mentioned transcription factor protein with halo-tag label thereto, and incubate with shaking on a mixer; S33 After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the equilibration buffer, shake on a mixer, take out and place on a magnetic rack, and discard the supernatant; S34 repeats the above steps S31-S33 to obtain a DNA fragment bound by a transcription factor; Treating TF-bound DNA with Tn5 transposase: Treating the DNA bound by the above-mentioned transcription factor with Tn5 transposase, and the Tn5 transposase binds to the region of the DNA fragment that is not protected by the transcription factor binding; treating the TF-bound DNA with Tn5 transposase includes the following steps: S41: remove the residual solution in the DNA fragment bound by the transcription factor, directly add the Tn5 transposase reaction solution, and incubate; S42 After incubation, place the tube on a magnetic rack at room temperature, aspirate the supernatant to rinse the tube wall so that all the magnetic beads sink to the bottom, discard the supernatant, immediately add the balancing buffer solution, shake on a mixer, take out and place on a magnetic rack until stratification, and discard the supernatant; S43 repeats the above steps S41 and S42 to obtain a product treated with Tn5 transposase; Construction of DNA library: Using PCR reaction system, constructing DNA library through PCR amplification; construction of DNA library includes the following steps: S52 sucks away the residual solution and directly adds the PCR reaction system; PCR reaction system: Tagged DNA 18µL; 2xHiFiPfu 20µL; N5primer 1µL, DAP-index, catalog number DAP-0103B; N7primer 1µL, DAP-index, catalog number DAP-0103B; The reaction program was: 68°C for 5 min; 98°C for 10 s; 60°C for 30 s for 18 cycles; 72°C for 30 s; 72°C for 2 min; S52 Measure the PCR product into a new centrifuge tube, add 1.05× magnetic beads into the centrifuge tube, pipette and mix, and place on the BLOCK plate at room temperature for 5-15 minutes; S53 Then place the centrifuge tube on the magnetic rack to separate layers, discard the supernatant, immediately add ethanol to wash, and after separation, discard the supernatant, immediately add ethanol, discard the supernatant, and absorb the remaining ethanol; Place on S54 magnetic rack, wait for the beads to dry, add ddH2O, remove the magnetic rack, and blow repeatedly; After S55 was allowed to stand, it was placed on a magnetic rack to stand and stratify, and the supernatant was transferred to a new EP tube and stored in the freezer; Sequencing and bioinformatics analysis: The products treated with Tn5 transposase were sequenced and analyzed using software to screen out transcription factor binding motifs.

2. The DAP-seq experimental method for building a fusion transposase library according to claim 1, characterized in that: The expression and enrichment of the transcription factor protein also includes preparing magnetic beads with halo-tag antibodies during the reaction of the wheat germ protein expression system: according to the amount of 10 µL of magnetic beads required for each protein sample, a corresponding volume of magnetic beads is taken out according to the number of samples, and the beads are washed on a magnetic rack with an equilibrium buffer in an EP tube, and finally PBS solution is added and evenly distributed in a corresponding number of EP tubes.

3. The DAP-seq experimental method for building a fusion transposase library according to claim 1, characterized in that: The sequencing and bioinformatics analysis include: 1) Data comparison: Use alignment software to align the data to the reference genome for comparison, filter out reads with low alignment quality values, use the unique alignment sequence obtained after alignment as the analysis object, analyze its coverage distribution on the reference genome, count the depth information of the genomic sites, and obtain the statistical results of the sequencing depth on the genome; 2) Information analysis: After sequencing, the DNA product is used to search for the binding sites between the target protein and DNA in the whole genome. The analysis software is used to perform peak scanning in the whole genome, and the peak information on the genome is analyzed to screen out peak-related genes. 3) Motif enrichment analysis based on transcription factor binding sites: within each peak, the number of Tn5 transposase insertion sites in the window was counted with a window size of 20 bp and a sliding step size of 5 bp, and adjacent windows were used as units; within each peak, 50 bp at both ends of the peak were excluded, and the window with the lowest insertion fragment depth was found as the candidate window containing the transcription factor binding site; for the top 500 peaks ranked by sequencing depth, candidate windows containing transcription factor binding sites were extracted and motif enrichment analysis was performed to screen protein-binding motifs.

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