A TAT-Tn5 transposase with improved nuclear entry efficiency, preparation and application in ATAC-seq

Abstract

A TAT-Tn5 transposase with improved nuclear entry efficiency, a preparation method, and an application in ATAC-seq, based on the existing E54K, L372P mutant Tn5 transposase, fused and expressed the puncture peptide TAT, expressed and purified in Escherichia coli, and assembled into a transposase body with a biotin-modified adapter sequence. The recombinant TAT-Tn5 transposase not only improves the efficiency of entering the cell nucleus, but also significantly improves the stability of the enzyme and reduces the preference for target DNA, providing a better choice for NGS sequencing.

Description

Technical Field

[0001] The present invention relates to the technical field of bioenzymes, and in particular to a TAT-Tn5 transposase capable of improving nuclear entry efficiency, and its preparation and application in ATAC-seq. Background Art

[0002] In chromatin accessibility sequencing (ATAC-seq), surfactants punch holes in the nuclear membrane, and then the Tn5 transposase enters the nucleus to randomly bind to and cut the DNA in the open region of chromatin, and inserts the adapter sequence at the cutting site. The success of ATAC-seq depends on the efficiency of Tn5 entering the nucleus and its transposition activity. However, different types of cells have significant differences in the composition of the plasma membrane and nuclear membrane. Too high a concentration of surfactant will cause the entire membrane structure to collapse, generating a large background signal. If the concentration is too low, the punching is not thorough, and it is difficult for the transposase to enter the nucleus, causing the experiment to fail. Therefore, it is best to perform gradient tests on different combinations of surfactants for each cell to screen the optimal surfactant combination and concentration. However, the optimization process will cause huge time and economic costs, greatly reducing the efficiency of the experiment. Therefore, it is urgent to develop an ATAC-seq method that does not rely on surfactant combinations and concentrations.

[0003] Frankel et al. first discovered in 1988 that the transactivator protein of human immunodeficiency virus HIV can penetrate the cell membrane and enter the cell. Further studies revealed that a segment of the amino acid sequence of the protein has the activity of penetrating the cell membrane, and it was subsequently named a penetrating peptide. Subsequent studies have discovered many other penetrating peptides. The chain length of these penetrating peptides generally does not exceed 30 amino acids and is positively charged. The amino acid sequence of the penetrating peptide is crucial to its ability to penetrate the membrane. Studies have found that the penetrating peptide TAT (YGRKKRRQRRR) with an 11-amino acid sequence has the strongest penetrating ability and low cytotoxicity. Subsequently, TAT was used to deliver different types of drug molecules, including nucleic acids, proteins, and small molecule drugs. In addition, TAT can increase the yield and solubility of heterologous proteins. TAT's strong ability to penetrate the membrane can carry the Tn5 transposase into the cell nucleus efficiently, which provides theoretical possibilities for the development of new methods. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a TAT-Tn5 transposase with improved nuclear import efficiency, and its preparation and application in ATAC-seq.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a TAT-Tn5 transposase with improved nuclear entry efficiency, the construction method of the TAT-Tn5 transposase comprising:

[0006] (1) Construction of expression vector:

[0007] The Tn5 transposase was fused with the puncture peptide to form the fusion protein TAT-Tn5, and the codons were optimized according to the amino acid sequence of TAT-Tn5. The optimized TAT-Tn5 coding sequence was subcloned into the pET28a-10xHis-Tev-SUMO vector by homologous recombination to obtain the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5, and the fusion expression of the tag SUMO was obtained;

[0008] (2) Induced expression of recombinant protein:

[0009] The TAT-Tn5 recombinant plasmid was transformed into the expression strain BL21 (DE3) for induced expression;

[0010] (3) Induction and purification of recombinant protein:

[0011] Collect the cells by centrifugation, crush them under high pressure and treat them by ultrasound; use a nickel column for preliminary purification, collect the components concentrated with the target protein, detect the purity of the target protein by SDS-PAGE, determine the concentration by Nanodrop, and calculate the total amount of the target protein; add ULP1 protease at a molar ratio of 1:20, dialyze overnight at 4°C to remove imidazole, and at the same time, ULP1 cuts the SUMO tag, and SDS-PAGE detects the cutting efficiency; the cutting product passes through the nickel column, the SUMO tag with the His tag is adsorbed by the nickel column, the target protein flows through, and the flow-through is collected; the collected flow-through passes through an anion exchange column for further purification; remove small molecular proteins and residual nucleic acids;

[0012] (4) Assembly of recombinant transposases:

[0013] The biotin-modified adapter primer was slowly annealed to form a double-stranded structure, and TAT-Tn5 and the double-stranded adapter were assembled at a molar ratio of 1:2 to form a transposase.

[0014] As a further improvement of the present invention: the construction of the expression vector comprises the following steps:

[0015] 1) The amino acid sequence of Tn5 transposase comes from plasmid pTBX1-Tn5, and the amino acid sequence of TAT comes from plasmid pTAT-Cre; the amino acid sequences of TAT and Tn5 are concatenated together for fusion expression, followed by codon optimization;

[0016] 2) Amplify the target gene, perform homologous recombination with the pET28a-10xHis-Tev-SUMO linear vector, select a single clone for Sanger sequencing verification, and obtain the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5.

[0017] As a further improvement of the present invention: in step 1), pTBX1-Tn5 (Addgene #60240) and pTAT-Cre (Addgene #35619) were codon optimized using the Gensmart codon optimization tool on the GenScript online website.

[0018] As a further improvement of the present invention: the amino acid sequence of TAT is shown in SEQ NO.1.

[0019] As a further improvement of the present invention: the base sequence of TAT is shown in SEQ NO.2.

[0020] As a further improvement of the present invention: comprising TAT-Tn5 transposase, the amino acid sequence of which is shown in SEQ ID NO.3.

[0021] As a further improvement of the present invention: comprising TAT-Tn5 transposase, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0022] As a further improvement of the present invention: including the induced expression of the recombinant protein, the induced expression of the recombinant protein comprises the following steps:

[0023] (1) Transform the recombinant plasmid into BL21(DE3), pick a single colony and inoculate it into 10 ml LB liquid culture, and culture it at 37°C and 220 rpm overnight;

[0024] (2) Take 1 mL of overnight culture and inoculate it into 1 LLB liquid medium, a total of 10 L, and culture at 37°C, 220 rpm until the OD600 absorbance is 0.6-0.8, then add IPTG at a final concentration of 0.3 mM and induce expression at 22°C, 220 rpm overnight;

[0025] (3) Centrifuge at 4000 rpm for 10 min at 4°C to collect the cells.

[0026] As a further improvement of the present invention: including the induced purification of the recombinant protein, the induced purification of the recombinant protein includes the following steps:

[0027] (1) Resuspend the cells in 100 mL of lysis buffer, which contains 10-100 mM Tris-HCl pH 7.0-9.0; 0.5-2 M NaCl; 5-40 mM Imidazole; 1-10% Glycerol.

[0028] (2) High-pressure crushing for 30 min and ultrasonic treatment for 20 min to fully crush the bacteria;

[0029] (3) Centrifuge at 12,000 g for 30 min at 4°C, transfer the supernatant to a new 50 mL centrifuge tube, load it onto a 5 mL nickel column at 5 mL / min, load the nickel column onto an AKTA purifier, rinse the nickel column with 100-300 mL Buffer A, and rinse with 5% Buffer B until the A280 detection line is balanced;

[0030] (4) SDS-PAGE run to detect the purity of the target protein;

[0031] (5) Mix the high-purity components together, measure the concentration using Nanodrop, add 5 mg of ULP1 protease for every 100 mg of target protein, mix well, dialyze overnight at 4°C, and use magnetic stirring to slowly rotate the dialysis bag;

[0032] (6) The dialyzed protein was loaded onto a 5 mL nickel column at 1 mL / min and the flow-through was collected;

[0033] (7) The protein flow-through was loaded onto a 5 mL Q column at 1 mL / min. The Q column was loaded onto an AKTA purifier. The nickel column was rinsed with 100-300 mL Buffer C and 5% Buffer D until the A280 detection line was balanced. A gradient of Buffer C and Buffer D was drawn. Within 30 min, the concentration of Buffer D increased from 5% to 100%, and the protein elution peak was collected at the same time.

[0034] (8) Mix the high-purity components together, add 4 volumes of saturated ammonium sulfate, let stand on ice for 30 min, centrifuge at 12,000 g for 30 min at 4°C, and discard the supernatant; resuspend the precipitate in 1 mL of dialysis buffer E; centrifuge at 12,000 g for 10 min at 4°C, and carefully load the supernatant onto the sample loop of the molecular sieve with a syringe. Set the molecular sieve flow rate to 0.5 mL / min, and collect the protein elution peak at the same time;

[0035] (9) Detect the purity of the target protein by SDS-PAGE;

[0036] (10) Pierce™ BCA Protein Quantitation Kit was used to detect the concentration of TAT-Tn5.

[0037] As a further improvement of the present invention:

[0038] Buffer A: 20 mM Tris-HCl pH 8.0; 1 M NaCl; 20 mM Imidazole; 5%Glycerol;

[0039] Buffer B: 20 ​​mM Tris-HCl pH 8.0; 1 M NaCl; 500 mM Imidazole; 5%Glycerol;

[0040] Buffer C: (20 mM Tris-HCl pH 8.0; 100 mM NaCl; 1 mM DTT; 5% Glycerol;

[0041] Buffer D: 20 mM Tris-HCl pH 8.0; 1000 mM NaCl; 1 mM DTT; 5% Glycerol;

[0042] Buffer E: 20 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM DTT; 5% Glycerol.

[0043] As a further improvement of the present invention: Pierce™ BCA Protein Assay Kit (Thermo Fisher Cat#23225).

[0044] As a further improvement of the present invention: comprising the assembly of a recombinant transposase, the assembly of the recombinant transposase comprises the following steps:

[0045] (1) The biotin-modified linker sequences Tn5ME-A, Tn5ME-B, and Tn5MERev were synthesized, and the sequence information is shown in SEQ NO.5.

[0046] (2) Tn5ME-A, Tn5ME-B, and Tn5MERev were dissolved in TE Buffer to a final concentration of 400 μM. Equal volumes of Tn5ME-A and Tn5MERev were slowly annealed to form adaptor A with a final concentration of 200 μM. Equal volumes of Tn5ME-B and Tn5MERev were slowly annealed to form adaptor B with a final concentration of 200 μM. Equal volumes of A and B were mixed to form adaptor A / B Mix with a final concentration of 200 μM.

[0047] (3) TAT-Tn5 and Adaptor Mix were mixed in equal molar proportions to form the following assembly system:

[0048] (4) Incubate at room temperature with rotation at 20 rpm for 1 h, divide into small portions, and store at -80°C.

[0049] The present invention also provides a use of the above-mentioned TAT-Tn5 transposase in ATAC-seq.

[0050] Compared with the prior art, the invention has the following beneficial effects: based on the existing E54K and L372P mutant Tn5 transposase, the patent fused and expressed the puncture peptide TAT, expressed and purified in Escherichia coli, and assembled into a transposase body with a biotin-modified linker sequence. The recombinant TAT-Tn5 transposase not only improves the efficiency of entering the cell nucleus, but also significantly improves the stability of the enzyme and reduces the preference for target DNA, providing a better choice for NGS sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a preparation flow chart of the present invention.

[0052] Figure 2 This is the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5 of the present invention. Recombinant plasmid map.

[0053] Figure 3 This is a graph showing the purity of the target protein detected by DS-PAGE gel running of the recombinant protein of the present invention.

[0054] Figure 4 Heat map and Metaplot analysis of the data volume of two biological replicates for the second-generation sequencing of the library. DETAILED DESCRIPTION

[0055] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0056] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0058] In the present application, the term "gene" refers to a nucleic acid fragment that expresses a specific protein or functional RNA molecule, which may include a regulatory sequence located before the coding sequence (5' non-coding region) and a regulatory sequence after the coding sequence (3' non-coding region).

[0059] In this application, the term "nucleic acid molecule" refers to a polymer of RNA or DNA, which is single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleic acid molecules in the form of DNA polymers can be composed of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0060] A TAT-Tn5 transposase with improved nuclear entry efficiency is disclosed. The Tn5 transposase is fused with a puncture peptide to form a fusion protein TAT-Tn5. Codon optimization is performed according to the amino acid sequence of TAT-Tn5 to prevent the appearance of secondary structures or rare codons, thereby clearing obstacles for high-efficiency expression in Escherichia coli. The optimized TAT-Tn5 coding sequence is subcloned into the pET28a-10xHis-Tev-SUMO vector by homologous recombination to obtain the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5; the fusion expression of the tag SUMO can enhance the soluble expression of the target protein. The TAT-Tn5 recombinant plasmid is transformed into the expression strain BL21 (DE3) for induced expression, the bacterial cells are collected by centrifugation, and high-pressure crushing and ultrasonic treatment are performed to fully lyse the bacterial cells and release the contents. Use nickel column for preliminary purification, collect the target protein concentrated components, detect the purity of the target protein by SDS-PAGE, determine the concentration by Nanodrop, and calculate the total amount of the target protein; add ULP1 protease at a molar ratio of 1:20, dialyze at 4℃ overnight to remove imidazole, and at the same time, ULP1 cuts the SUMO tag, and SDS-PAGE detects the cutting efficiency. The cleavage product passes through the nickel column, the SUMO tag with Histag is adsorbed by the nickel column, the target protein flows through, and the flow-through is collected. The collected flow was further purified by passing through an anion exchange column, eluted with sodium chloride gradient, and the eluted sample was collected and concentrated to 2 ml; fine purification was performed by molecular sieve Superdex ®200 Increase 10 / 300 to further remove small molecular proteins and residual nucleic acids, and the concentration of recombinant TAT-Tn5 was determined by BCA kit; the biotin-modified adapter primer was slowly annealed to form a double-stranded structure, and TAT-Tn5 and the double-stranded adapter were assembled at a molar ratio of 1:2 to form a transposase; ATAC-seq library was constructed, the library was sequenced by next-generation sequencing, and the library quality was analyzed by bioinformatics.

[0061] Implementation Cases:

[0062] 1. Construction of expression vector:

[0063] (1) The amino acid sequence of Tn5 transposase was derived from plasmid pTBX1-Tn5 (Addgene #60240), and the amino acid sequence of TAT was derived from plasmid pTAT-Cre (Addgene #35619). The amino acid sequences of TAT and Tn5 were concatenated for fusion expression, and then codon optimization was performed using the Gensmart codon optimization tool on the GenScript online website. The amino acid sequence of the final puncture peptide TAT is shown in SEQ NO.1, and the corresponding base sequence is shown in SEQ NO.2; the amino acid sequence of TAT-Tn5 is shown in SEQ NO.3, and the corresponding base sequence is shown in SEQ NO.4.

[0064] (2) Amplify the target gene, perform homologous recombination with the pET28a-10xHis-Tev-SUMO linear vector, select a single clone for Sanger sequencing verification, and obtain the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5. The recombinant plasmid map is shown in Figure 1 As shown, the complete reading frame sequence is shown in SEQ NO. 2.

[0065] 2. Induced expression of recombinant protein:

[0066] (1) Transform the recombinant plasmid into BL21(DE3), pick a single colony and inoculate it into 10 ml LB liquid culture, and culture it overnight at 37°C and 220 rpm.

[0067] (2) Take 1 mL of overnight culture and inoculate it into 1 LLB liquid medium, for a total of 10 L. Cultivate at 37°C, 220 rpm until the OD600 absorbance reaches 0.6-0.8. Add IPTG at a final concentration of 0.3 mM and induce expression at 22°C, 220 rpm overnight.

[0068] (3) Centrifuge at 4000 rpm for 10 min at 4°C to collect the cells.

[0069] 3. Induction and purification of recombinant protein:

[0070] (1) Resuspend the cells in 100 mL of lysis buffer, which contains 10-100 mM Tris-HCl pH 7.0-9.0; 0.5-2 M NaCl; 5-40 mM Imidazole; 1-10% Glycerol. The preferred buffer is 20 mMTris-HCl pH 8.0; 1.5 M NaCl; 20 mM Imidazole; 5% Glycerol.

[0071] (2) High-pressure crushing treatment for 30 min and ultrasonic treatment for 20 min were performed to fully crush the bacteria.

[0072] (3) Centrifuge at 12,000 g for 30 min at 4°C. Transfer the supernatant to a new 50-mL centrifuge tube and load it onto a 5-mL nickel column at 5 mL / min. Load the nickel column into an AKTA purifier and rinse the nickel column with 100-300 mL of Buffer A (20 mM Tris-HCl pH 8.0; 1 M NaCl; 20 mM Imidazole; 5% Glycerol). Rinse the nickel column with 5% Buffer B (20 mM Tris-HCl pH8.0; 1 M NaCl; 500 mM Imidazole; 5% Glycerol) until the A280 detection line is balanced. Create a gradient between Buffer A and Buffer B. Increase the concentration of Buffer B from 5% to 100% within 30 min. Collect the protein elution peak at the same time.

[0073] (4) SDS-PAGE run to detect the purity of the target protein, such as Figure 3 shown.

[0074] (5) Mix the high-purity components together, measure the concentration using Nanodrop, add 5 mg of ULP1 protease for every 100 mg of target protein, mix well, dialyze overnight at 4°C, and use magnetic stirring to slowly rotate the dialysis bag. The dialysate composition is 20 mM Tris-HCl pH 8.0; 100 mM NaCl; 10 mM Imidazole; 10% Glycerol.

[0075] (6) The dialyzed protein was loaded onto a 5 mL nickel column at 1 mL / min and the flow-through was collected.

[0076] (7) The protein flow-through was loaded onto a 5 mL Q column at 1 mL / min. The Q column was loaded onto an AKTA purifier. The nickel column was rinsed with 100-300 mL Buffer C (20 mM Tris-HCl pH 8.0; 100 mM NaCl; 1 mM DTT; 5% Glycerol). The nickel column was rinsed with 5% Buffer D (20 mM Tris-HCl pH 8.0; 1000 mM NaCl; 1 mM DTT; 5% Glycerol) until the A280 detection line was balanced. A gradient of Buffer C and Buffer D was drawn. The concentration of Buffer D was increased from 5% to 100% within 30 min. The protein elution peak was collected at the same time.

[0077] (8) Mix the high-purity fractions together, add 4 times the volume of saturated ammonium sulfate, let stand on ice for 30 min, centrifuge at 4°C, 12000 g for 30 min, and discard the supernatant. Resuspend the precipitate in 1 mL of dialysis buffer Buffer E (20 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM DTT; 5% Glycerol). Centrifuge at 4°C, 12000 g for 10 min, carefully load the supernatant onto the molecular sieve sample loop with a syringe, set the molecular sieve flow rate to 0.5 mL / min, and collect the protein elution peak at the same time.

[0078] (9) SDS-PAGE to detect the purity of the target protein, such as Figure 3 shown.

[0079] (10) The concentration of TAT-Tn5 was detected using Pierce™ BCA Protein Assay Kit (Thermo Fisher Cat #23225).

[0080] 4. Assembly of recombinant transposases:

[0081] (1) The biotin-modified linker sequences Tn5ME-A, Tn5ME-B, and Tn5MERev were synthesized, and the sequence information is shown in SEQ NO.5.

[0082] (2) Tn5ME-A, Tn5ME-B, and Tn5MERev were dissolved in TE Buffer to a final concentration of 400 μM. Equal volumes of Tn5ME-A and Tn5MERev were slowly annealed to form adaptor A with a final concentration of 200 μM. Equal volumes of Tn5ME-B and Tn5MERev were slowly annealed to form adaptor B with a final concentration of 200 μM. Equal volumes of A and B were mixed to form adaptor A / B Mix with a final concentration of 200 μM.

[0083] (3) TAT-Tn5 and Adaptor Mix were mixed in equal molar proportions to form the following assembly system:

[0084] (4) Incubate at room temperature with rotation at 20 rpm for 1 h, divide into small portions, and store at -80°C.

[0085] 5. ATAC-seq activity test:

[0086] (1) Select K562 suspension cells for testing. Centrifuge 10 ml of cultured human ovarian cancer cell Skov3 at 4°C, 500 g for 5 min and discard the supernatant. Add 1 ml of 1× PBS to resuspend the cells and centrifuge at 4°C, 500 g for 5 min. Collect the cell pellet and resuspend it in 1 ml of DPBS.

[0087] (2) Count the cells and adjust the cell concentration to 106 / mL. Take 50 μL of the cell suspension (about 5000 cells), add 4 μL of DNase I, mix by pipetting, and incubate at 37°C for 30 min.

[0088] (3) Centrifuge at 4°C, 500 g for 5 min, discard the supernatant, add 200 μL 1× PBS to resuspend the cells, centrifuge at 4°C, 500 g for 5 min, and collect the cell pellet (to remove DNase I).

[0089] (4) Add 100 μL of pre-cooled RSB1 (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% NP-40; 0.1% Tween-20; 0.01% Digitonin) to change the permeability of the cell membrane, pipette to mix, and incubate on ice for 3 min.

[0090] (5) Add 1 ml of pre-cooled RSB2 (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20) to terminate the RSB1 reaction, invert to mix, centrifuge at 4°C, 500 g for 5 min, discard the supernatant, and collect the cell pellet.

[0091] (6) Configure Tn5 reaction system

[0092] Table 1 Tn5 reaction and volume statistics

[0093] Reagents Volume μL 1× PBS 16.5 μL 5×Tagment Buffer 10 μL 1% Digitonin 0.5 μL 10% Tween-20 0.5 μL TAT-Tn5 enzyme (with embedded adapter) 5 μL H2O 17.5 μL Total 50 μL

[0094] (7) Use a 1000μL pipette tip to remove 1ml of liquid from the upper layer, and a 10μL pipette tip to remove 100μL of liquid (try to remove as much liquid as possible from the tube, leaving the cells at the bottom). Add the prepared mix and incubate at 37℃, 1000rpm for 30min (you can quickly freeze it after incubation).

[0095] (8) DNA recovery

[0096] 1) Add 1 μL 10% SDS, flick to mix, and incubate at 55° for 10 min.

[0097] 2) Collect the liquid in the tube cap by centrifugation, blow the magnetic beads with a pipette, and place them on the magnetic stand.

[0098] 3) Take 2 μL of streptavidin magnetic beads, wash twice with PBS on a magnetic rack, resuspend in 2 μL PBS, and add the above 50 μL supernatant.

[0099] 4) Incubate at room temperature with rotation or shaking for 20 min.

[0100] 5) Rinse three times with 200 μL PBS and resuspend the magnetic beads with 20 μL PBS.

[0101] (9) Library amplification

[0102] 1) Configure the amplification system

[0103] Table 2 Statistics of reagents and volumes of amplification system

[0104] Reagents volume C1 magnetic beads recovered in the previous step 20 μL 10 μM Forward Primer 2.5 μL 10 μM Reverse Primer 2.5 μL 2×KAPA HiFi HotStart ReadyMix 25 μL Total 50 μL

[0105] 2) Reaction procedure

[0106] Table 3 Temperature control cycle table of hot cover temperature in reaction program

[0107]

[0108] 3) Library purification quality inspection

[0109] a. Take out the DNA magnetic beads and equilibrate at room temperature for 30 minutes;

[0110] b. Add 50 μL DNA magnetic beads at a ratio of 1:1, shake and mix, and leave at room temperature for 5 minutes;

[0111] c. Place on a magnetic rack and discard the supernatant after the solution becomes clear;

[0112] d. Add 200 μL 80% ethanol to wash the beads, discard the ethanol, and repeat twice;

[0113] e. After the magnetic beads are dried, add 20 μL of DEPC water, shake and mix, and measure the DNA concentration after leaving it for 2 minutes;

[0114] f. Take the library dilution solution to 1-2 ng / μL for fragment quality inspection, as shown in the following table. The fragments are concentrated in the length of 1~2 nucleosomes, and the fragment distribution is better.

[0115] Table 4 Comparison of sequencing data from parallel experiments

[0116] project The number of sequencing sequences after sequencing data filtering The number of sequences that can be mapped to the genome The percentage of sequencing sequences that can be located on the genome The number of sequence reads with unique alignments on the reference sequence The percentage of sequenced sequences with unique alignment positions on the reference sequence Skov3-1 17840646 14386256 95.78% 12796044 71.70% Skov3-2 16780993 13530434 95.91% 12057105 71.85%

[0117] 6. Bioinformatics analysis:

[0118] The library was sequenced for the next generation. The data volume of two biological replicates was about 20G, and the detected reads were about 17M. Among them, the proportion that could be mapped to the genome was 95%, and the proportion of unique reads was 75%. The data quality was good. The subsequent heat map and Metaplot analysis revealed that Figure 4As shown, the sequencing fragments are obviously concentrated at the transcription start site, and the background signal is weak, indicating that TAT-Tn5 can be used in ATAC-seq experiments.

[0119] In summary, after reading the present invention document, ordinary technicians in this field can make various other corresponding transformation schemes based on the technical scheme and technical concept of the present invention without creative mental work, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a TAT-Tn5 transposase with improved nuclear entry efficiency, characterized in that: The construction method of the TAT-Tn5 transposase comprises: (1) Construction of expression vector: The Tn5 transposase was fused with the puncture peptide to form the fusion protein TAT-Tn5, and the codons were optimized according to the amino acid sequence of TAT-Tn5. The optimized TAT-Tn5 coding sequence was subcloned into the pET28a-10xHis-Tev-SUMO vector by homologous recombination to obtain the recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5; The nucleotide sequence of the fusion protein TAT-Tn5 transposase is shown in SEQ ID NO.4; (2) Induced expression of recombinant protein: The recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5 was transformed into BL21 (DE3) for inducible expression: The inducing expression comprises the following steps: [1] The recombinant plasmid pET28a-10 His-Tev-SUMO-TAT-Tn5 was transformed into BL21 (DE3), and a single colony was picked and inoculated into 10 ml LB liquid culture, and cultured overnight at 37°C and 220 rpm; [2] Take 1 mL of overnight culture and inoculate 1 L of liquid medium, a total of 10 L, and culture at 37°C, 220 rpm until the OD600 absorbance is 0.6-0.

8. Add IPTG at a final concentration of 0.3 mM and induce expression at 22°C, 220 rpm overnight. [3] Centrifuge at 4000 rpm for 10 min at 4°C to collect the cells; (3) Induction and purification of recombinant protein: The collected bacteria were crushed under high pressure and treated with ultrasound; a nickel column was used for preliminary purification, and the components concentrated in the target protein were collected. The purity of the target protein was detected by SDS-PAGE, and the concentration was determined by Nanodrop to calculate the total amount of the target protein; ULP1 protease was added at a molar ratio of 1:20, and imidazole was removed by dialysis at 4°C overnight. At the same time, ULP1 cleaved the SUMO tag, and the cleavage efficiency was detected by SDS-PAGE; the cleavage product passed through the nickel column, the SUMO tag with the His tag was adsorbed by the nickel column, the target protein flowed through, and the flow-through was collected; the collected flow-through was further purified by passing through an anion exchange column; small molecular proteins and residual nucleic acids were removed to obtain TAT-Tn5; (4) Assembly of recombinant transposases: The biotin-modified linker primer was slowly annealed to form a double-stranded linker, and TAT-Tn5 and the double-stranded linker were assembled at a molar ratio of 1:2 to form TAT-Tn5 transposase.

2. Use of the TAT-Tn5 transposase prepared by the preparation method according to claim 1 in ATAC-seq.

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