CN116891532A - Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA - Google Patents

Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA Download PDF

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CN116891532A
CN116891532A CN202310316670.3A CN202310316670A CN116891532A CN 116891532 A CN116891532 A CN 116891532A CN 202310316670 A CN202310316670 A CN 202310316670A CN 116891532 A CN116891532 A CN 116891532A
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transposase
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易文洋
曹林
聂俊伟
瞿志鹏
江明扬
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Nanjing Novozan Biotechnology Co ltd
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Abstract

The invention provides a biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA.

Description

Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA
Technical Field
The present invention relates to the field of biotechnology. The invention relates to a secondary antibody-transposase complex based on biotin-avidin and a mixture thereof. The invention relates in particular to the use of said complexes or mixtures thereof for detecting protein-DNA interactions. The invention particularly relates to a method capable of improving the interaction accuracy of detection proteins and DNA, effectively reducing the background and improving the output of real data and application thereof.
Background
Chromatin immunoprecipitation (ChIP) is a widely used method for studying protein-DNA interactions, typically for the study of transcription factor binding sites or histone-specific modification sites. Combining ChIP with large-scale parallel DNA sequencing techniques allows researchers to precisely map the global DNA binding site of a protein of interest. The basic flow of ChIP is: (1) crosslinking: fixing tissues or cells by formaldehyde to tightly combine DNA and protein; (2) fragmenting: this process disrupts chromatin, ultimately obtaining a DNA fragment protein complex for ChIP analysis; (3) chromatin immunoprecipitation: binding the target protein-DNA complex by adding an antibody against the protein of interest; (4) DNA recovery and purification: the recovered DNA fragments were purified and enriched, and DNA sequence information (doi: 10.1038/nrg 2641) of specific binding of the target protein was analyzed by downstream detection techniques (quantitative PCR, gene chip, sequencing, etc.) (FIG. 1). However, because ChIP technology requires formaldehyde crosslinking of tissues or cells followed by DNA fragmentation, this technology requires extremely high initial inputs of cells (millions), and there is a high likelihood that experimental results will be false positive because of excessive formaldehyde crosslinking.
To remedy the technical defects of ChIP, researchers have developed the CUT & Run (Cleavage Under Targets & Release Using Nuclease) and CUT & Tag (Cleavage UnderTargets & Tag) technologies (DOI: 10.7554/ehife.21856.001; doi.org/10.1038/s 41467-019-09982-5) through continuous updating and optimization. CUT & Tag and CUT & Run are techniques for studying protein-DNA interactions in living cells of a living organism by antibody enrichment of DNA near the protein of interest for Tn5 transposase or MNase nuclease specific cleavage, followed by library construction and sequencing of the labeled DNA after cleavage to map the global DNA binding site for the protein of interest (fig. 2). The technology alleviates the disadvantages of false positives and high cell input of the CHIP technology to a certain extent because crosslinking and physical disruption of DNA are not required.
Although the CUT & Tag and CUT & Run technologies are significantly improved over ChIP, the CUT & Tag and CUT & Run dependent PA/PG-Tn5/Mnase fusion proteins bind to the secondary antibody Fc segment and then Tn5/Mnase is brought into the vicinity of the target protein region for DNA cleavage. This process is limited on the one hand by the ability of the PA/PG protein to bind to the Fc fragment of the secondary antibody and on the other hand by the poor quantitative relationship between the PA/PG-Tn5/MNAse fusion protein and the secondary antibody, which is highly likely to result in excessive incubation of the PA/PG-Tn5/MNAse fusion protein. The non-specifically bound PA/PG-Tn5/Mnase fusion protein will nonspecifically cleave the chromatin open region during CUT & Tag/CUT & Run experiments, thus generating a large number of TSS (Transcription Start Sites) region false positive signals (FIG. 3), which will greatly interfere with the authenticity of the CUT & Tag/CUT & Run experimental data. Moreover, enrichment of false positive signals in the TSS region can also lead to low real signal data duty ratio at the same sequencing depth, and the output of effective data is reduced.
Summary of The Invention
Object of the Invention
1. Solves the false positive problem in CUT & Tag and CUT & Run experiments.
2. The CUT & Tag and CUT & Run experimental procedures are optimized, washing steps are reduced, and amplification can be directly carried out without template extraction, so that the experimental time is shortened.
3. The innovative construction mode of the compound can be used for carrying out in vitro tight combination of eukaryotic proteins and prokaryotic proteins.
Disclosure of Invention
1. The invention constructs the compound of different secondary antibodies and Tn5/MNAse based on biotin-avidin binding pairs, the compound can effectively reduce the quantity of free Tn5 and MNAse, and in CUT & Tag and CUT & Run experiments, primary antibody incubation, secondary antibody incubation, protein A (PA)/Protein G (PG) -Tn5/MNAse incubation are optimized as primary antibody incubation, and secondary antibody-T5/MNAse compound incubation. The experimental operation flow is reduced, tn5/MNase is firmly bound through the secondary antibody-T5/MNase complex, free Tn5/MNase exposure in the original CUT & Tag and CUT & Run experiments is reduced, and false positive signals of a negative control TSS region are obviously reduced.
2. Since the secondary antibody is generally derived from mouse, rabbit, goat and other species, and Tn5 enzyme and the like are derived from escherichia coli, the difficulty in constructing fusion protein is great, and after the secondary antibody-Tn 5/Mnase fusion protein is expressed in escherichia coli, the secondary antibody has low binding efficiency, which is possibly related to the difference between eukaryotic and prokaryotic post-transcriptional modification. To overcome the problem that secondary antibodies and Tn5 host sources are different, so that secondary antibody-Tn 5/Mnase fusion proteins with functional activity cannot be obtained, the secondary antibodies are innovatively subjected to Biotin (BT) modification, and then incubated with avidin, particularly Streptavidin (SA) -Tn5/Mnase fusion proteins in vitro (or the secondary antibodies are subjected to BT modification and then are coupled with SA, and simultaneously Tn5/Mnase is subjected to BT modification, and then the secondary antibodies and the streptavidin are incubated in vitro). SA BT has a binding capacity of Kd=10-15, which is at least ten thousand times higher than the highest antigen-antibody binding capacity. And 4 BT molecules can be combined with one SA protein, and the Tn5/Mnase is ensured to be captured by the secondary antibody mostly through the ratio control of SA to BT, so that the functional secondary antibody-BT: SA-Tn5/Mnase complex and the functional secondary antibody-BT: SA: BT-Tn5/Mnase complex are achieved.
3. After CUT & Tag enzyme digestion reaction, adding a stop solution and amplification Mix with a special sequence connector, and directly constructing a library without purification. The step can reduce the experimental operation flow, complete all library building experiments in one EP pipe, and is beneficial to the construction of subsequent automatic platforms.
Advantageous effects
1. The secondary antibody-BT: SA: BT-Tn5/Mnase and secondary antibody-BT: SA-Tn5/Mnase complex enable Tn5/Mnase to be firmly grabbed by the secondary antibody, and the combination degree of the Tn5/Mnase enzyme and the secondary antibody is far stronger than that of PA/PG-Tn5 and the secondary antibody, so that the background of negative control in the CUT & Tag and CUT & Run experiment process is obviously reduced, and false positives in the CUT & Tag and CUT & Run technology are greatly reduced.
2. The overall effective data was more than the experimental data signal enrichment was significantly increased (fig. 5).
3. The time of the overall CUT & Tag and CUT & Run experiment is reduced by 6 hours, and the overall efficiency of the experiment is improved.
4. The use of SA modification and BT modification to construct complexes makes it possible to integrate eukaryotic and prokaryotic proteins and retain their original activity. In particular, immune related proteins involve VDJ rearrangement and abundant post-transcriptional modifications, and direct expression of the related proteins in a prokaryotic system is not immunologically active. The SA modification can enable the immune related protein to be combined with other prokaryotic proteins after being expressed in a primary host, so that the eukaryotic protein-prokaryotic protein complex with biological activity is obtained, the eukaryotic protein-prokaryotic protein complex shows accurate target point positioning in a CUT & Tag experiment, and the target gene TSS region has obvious signal enrichment (figure 5).
Description of the embodiments
The present invention relates to the following embodiments.
1. A biotin-avidin-based secondary antibody transposase complex represented by the formula:
transposase-avidin (biotin-second antibody) n Wherein n is 1, 2, 3 or 4.
2. The complex of embodiment 1, wherein the avidin is streptavidin.
3. The complex of embodiment 1, wherein the transposase is a Tn5 transposase.
4. A mixture of complexes according to any of embodiments 1-3, represented by the formula:
transposase-avidin (biotin-second antibody) p Wherein p is greater than 1 and less than 4.
5. A biotin-avidin-based secondary antibody transposase complex represented by the formula:
(transposase-biotin) m Avidin (biotin-second antibody) n Wherein m and n are each independently 1, 2 or 3,
and m+n is less than or equal to 4.
6. The complex of embodiment 5, wherein the avidin is streptavidin.
7. The complex of embodiment 5, wherein the transposase is a Tn5 transposase.
8. The complex of embodiment 5 wherein the biotin is attached to an oligonucleotide of the transposome, optionally the oligonucleotide is a partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3'(SEQ ID NO：
12 And 5' -TCGT BT CGGCAGCGTCAGATGTGTAT BT AAGAGACAG-3 '(SEQ ID NO: 195) and/or partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3 '(SEQ ID NO: 12) and 5' -GTCTCGTGGGCT BT CGGAGATGTGTAT BT AAGAGACAG-3
(SEQ ID NO: 297) or the oligonucleotide is a partially paired 5' -phos-CT BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -TCGTCGGCAGCGTCAGATGTGT BT AT BT AAGAGACAG-3′(SEQ ID NO：
215 5' -phos-CT) and/or partial pairing BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -GTCT BT CGTGGGCT BT CGGAGATGTGTATAAGAGACAG-3′
(SEQ ID NO：317)，T BT Representing biotinylated T.
9. A mixture of complexes according to any of embodiments 5-8, represented by the formula:
(transposase-biotin) q Avidin (biotin-second antibody) p Wherein p and q are each greater than or equal to 1 and less than or equal to 3, and q+p is less than or equal to 4.
10. A method of detecting interaction of a cell with DNA at a target protein, comprising:
(1) Incubating the cells with a primary antibody directed against the target protein;
(2) Incubating the cells with the complex of any of embodiments 1-3 and 5-8 or the mixture of embodiments 4 or 9, wherein the secondary antibody in the complex is a secondary antibody specific for the primary antibody and allowing the transposase to act on the genome of the cells;
(3) Amplifying the cellular DNA;
(4) Purifying the amplified DNA; and is combined with
(5) The amplified DNA was sequenced.
Drawings
Fig. 1: a Chip-Seq technical schematic diagram;
fig. 2: a CUT & Tag experimental schematic;
fig. 3: the conventional CUT & Tag experimental IgG negative control has obvious signal enrichment in the TSS region;
fig. 4: signal comparison of three groups of negative control libraries (IgG+secondary antibody+pGTn5, igG+secondary antibody: pG-Tn5 and IgG+secondary antibody-BT: SA-Tn 5) in example 2;
fig. 5: comparison of true signals for six sets of samples in example 2;
fig. 6: comparison of the reference library (primary+secondary+pGTn5, primary+secondary: pG-Tn5) and test library (primary+secondary-BT: SA-Tn 5) distributions in example 2;
fig. 7: a negative control (IgG+secondary antibody-BT: SA: BT-Tn5-1, igG+secondary antibody-BT: SA: BT-Tn 5-2) signal pattern in example 3;
fig. 8: comparison of true signals for eight sets of samples in example 3;
fig. 9: four library profiles in example 3.
Detailed Description
In one aspect, the invention provides a biotin-avidin-based secondary antibody transposase complex represented by the formula:
transposase-avidin (biotin-second antibody) n Wherein n is 1, 2, 3 or 4.n is the number of secondary antibody molecules loaded by the transposase molecules.
In one embodiment, the avidin is streptavidin or neutravidin. In one embodiment, the transposase is a Tn5 transposase.
In one aspect, the present invention provides a mixture of the above complexes, represented by the formula:
transposase-parentSynthctine (biotin-second antibody) p Wherein p is the average biotin-secondary antibody load of transposase-avidin, greater than 1 and less than 4.p is the average number of secondary antibody molecules loaded by the transposase molecules in the mixture.
In one aspect, the present invention provides a method of preparing the above-described complex or mixture comprising: expressing a transposase-avidin fusion protein in E.coli;
a biotinylated secondary antibody; and mixing the transposase-avidin fusion protein with the biotinylated secondary antibody in a molar ratio of 1:n'.
In one embodiment, n' =n. In one embodiment, n' > n. In one embodiment, n' =p. In one embodiment, n' > p. In one embodiment, n '>4, e.g., n' =5. In one embodiment, the molar ratio of transposase-avidin fusion protein to biotinylated secondary antibody is such that no free transposase-avidin (i.e., not bound to biotinylated secondary antibody) is present. In one embodiment, the molar ratio of the transposase-avidin fusion protein to the biotinylated secondary antibody is such that the avidin in the transposase-avidin is saturated with biotin in the biotin-secondary antibody.
In one aspect, the invention provides a biotin-avidin-based secondary antibody transposase complex represented by the formula:
(transposase-biotin) m Avidin (biotin-second antibody) n Wherein m and n are each independently 1, 2 or 3, and m+n is less than or equal to 4, e.g., m+n=2, 3 or 4. For example, m=1, n=1, 2 or 3; m=2, n=1 or 2; m=3, n=1. As another example, n=1, m=1, 2 or 3; n=2, m=1 or 2; n=3, m=1. m is the number of transposase molecules loaded by avidin molecules, and n is the number of secondary antibody molecules loaded by avidin molecules.
In one embodiment, the avidin is streptavidin or neutravidin. In one embodiment, the transposase is a Tn5 transposase. In one embodiment, the biotin is attached to an oligonucleotide of a transposome. In one embodiment, the transposase molecule (or related oligonucleotide) is loaded with oneOr a plurality of biotin molecules, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more. In one embodiment, the oligonucleotide is as shown in CTGTCTCTTTATACACATCT (SEQ ID NO: 1). In one embodiment, the oligonucleotide is as set forth in SEQ ID NO: 4-182. In one embodiment, the oligonucleotide is as shown in TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 2). In one embodiment, the oligonucleotide is as set forth in SEQ ID NO: 183-284. In one embodiment, the oligonucleotide is as shown in GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 3). In one embodiment, the oligonucleotide is as set forth in SEQ ID NO: 285-464. In one embodiment, SEQ ID NO:1 and SEQ ID NO: part 2 pairs. In one embodiment, SEQ ID NO:4182 and any one of SEQ ID NOs: 183-284. In one embodiment, SEQ ID NO:1 and SEQ ID NO:3 parts pairing. In one embodiment, SEQ ID NO:4-182 and SEQ ID NO: 285-464. In one embodiment, the transposase is paired with a portion of SEQ ID NO:1 and SEQ ID NO:2 compounding. In one embodiment, the transposase is paired with a portion of SEQ ID NO:4-182 and SEQ ID NO: 183-284. In one embodiment, the transposase is paired with a portion of SEQ ID NO:1 and SEQ ID NO: and 3, compounding. In one embodiment, the transposase is paired with a portion of SEQ ID NO:4-182 and SEQ ID NO: 285-464. In one embodiment, the transposase is paired with a portion of SEQ ID NO:1 and SEQ ID NO:2 and with a partial pairing of SEQ ID NO:1 and SEQ ID NO: and 3, compounding. In one embodiment, the transposase is paired with a portion of SEQ ID NO:4-182 and SEQ ID NO:183-284 and paired with a portion of SEQ ID NO:4-182 and SEQ ID NO: 285-464. In one embodiment, SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 are each independently attached to one or more biotin molecules, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more. In one embodiment, the oligonucleotide is a partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3 '(SEQ ID NO: 12) and 5' -TCGT BT CGGCAGCGTCAGATGTGTAT BT AAGAGACAG-3 '(SEQ ID NO: 195) and/or partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3 '(SEQ ID NO: 12) and 5' -GTCTCGTGGGCT BT CGGAGATGTGTAT BT AAGAGACAG-3 (SEQ ID NO: 297) or alternatively the oligonucleotide is a partially paired 5' -phos-CT BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -TCGTCGGCAGCGTCAGATGTGT BT AT BT AAGAGACAG-3 '(SEQ ID NO: 215) and/or partially paired 5' -phos-CT BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -GTCT BT CGTGGGCT BT CGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：317)，T BT Representing biotinylated T.
In one aspect, the present invention provides a mixture of the above complexes, represented by the formula:
(transposase-biotin) q Avidin (biotin-second antibody) p Wherein p and q are each greater than or equal to 1 and less than or equal to 3, and q+p is less than or equal to 4, e.g., q+p≡2, 3 or 4. For example, q≡1, p≡1, 2 or 3; q is equal to 2, p is equal to 1 or 2; q is approximately equal to 3 and p is approximately equal to 1. For another example, p.apprxeq.apprxeq.apprxeq.1, 2 or 3; p.about.2, q.about.1 or 2; p.apprxeq.apprxeq.apprxeq.apprxeq.apprxeq.apprxeq.apprxeq.1. q is the average number of transposase molecules loaded with avidin molecules in the mixture, and p is the average number of secondary antibody molecules loaded with avidin molecules in the mixture.
In one aspect, the present invention provides a method of preparing the above-described complex or mixture comprising: a biotinylated secondary antibody;
a biotinylated transposase;
mixing avidin and biotinylated secondary antibody in the molar ratio of 1 to n' to obtain intermediate;
and
The intermediate was mixed with biotinylated transposase in a molar ratio of 1:m'.
In one aspect, the present invention provides a method of preparing the above-described complex or mixture comprising: a biotinylated secondary antibody;
a biotinylated transposase;
mixing avidin and biotinylated transposase in a molar ratio of 1:m' to obtain an intermediate;
and
The intermediate was mixed with biotinylated secondary antibody in a molar ratio of 1:n'.
In one aspect, the present invention provides a method of preparing the above-described complex or mixture comprising: a biotinylated secondary antibody;
a biotinylated transposase;
and mixing the biotinylated transposase, avidin and the biotinylated secondary antibody in a molar ratio of m ':1:n'.
In one embodiment, n' =n. In one embodiment, n' > n. In one embodiment, n' =p. In one embodiment, n' > p. In one embodiment, n '<4, e.g., n' <3, e.g., n '<2, e.g., n' =1, 2, or 3. In one embodiment, n' >4. In one embodiment, m' =m. In one embodiment, m' > m. In one embodiment, m' =q. In one embodiment, m' > q. In one embodiment, m '<4, e.g., m' <3, e.g., m '<2, e.g., m' =1, 2, or 3. In one embodiment, m' >4. In one embodiment, where n ' =1, m ' +.3, e.g., m ' <3. In one embodiment, where n ' =2, m ' +.2, e.g., m ' <2. In one embodiment, in the case where n ' =3, m ' +.1, e.g., m ' <1. In one embodiment, where m ' =1, n ' +.3, e.g., n ' <3. In one embodiment, where m ' =2, n ' +.2, e.g., n ' <2. In one embodiment, where m ' =3, n ' +.1, e.g., n ' <1. In one embodiment, n '>4 and m' >4. In one embodiment, the molar ratio is such that free (i.e., non-biotinylated secondary antibody bound) transposase is not present.
In one embodiment, the values of the present invention include errors of + -20%, + -10%, or + -5%.
In one aspect, the invention provides a method of detecting interaction of a cell with DNA at a target protein, comprising:
(1) Incubating the cells with a primary antibody directed against the target protein;
(2) Incubating a cell with the complex or mixture, wherein the secondary antibody in the complex is a secondary antibody specific for the primary antibody and allowing the transposase to act on the genome of the cell;
(3) Amplifying the cellular DNA;
(4) Purifying the amplified DNA; and is combined with
(5) The amplified DNA was sequenced.
In one embodiment, the method of the invention directly amplifies cellular DNA without extracting and purifying the cellular DNA.
In one embodiment of the present invention, in one embodiment, the target protein is AAF, ab1, ADA2, ADA-NF1, AF-1, AFP1, ahR, AIIN3, ALL-1, alpha-CBF, alpha-CP 1, alpha-CP 2a, alpha-CP 2B, alpha Ho, alpha H2-alpha FB, alx-4, aMEF-2, AML1a, AML1B, AML1C, AML1 delta N, AML2, AML3a, AML3B AMY-1L, A-Myb, ANF, AP-1, AP-2α A, AP-2α B, AP-2β, AP-2γ, AP-3 (1), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, arnt (774M form), ARP-1, ATBF1-A, ATBF-B, ATF, ATF-1, ATF-2, ATF-3, ATF-3δZIP, ATF-a, ATF-aδ, ATPF1, barhl2, arnt Barx1, barx2, bcl-3, BCL-6, BD73, beta-catenin, bin1, B-Myb, BP1, BP2, brahma, BRCA1, brn-3a, brn-3B, brn-4, BTEB2, B-TFIID, C/EBPα, C/EBPβ, C/EBPδ, CACC binding factor, cart-1, CBF (4), CBF (5), CBP, CCAAT-binding factor, CCMT-binding factor, CCF, CCG1, CCK-1a, CCK-1B, CD28RC, cdk2, cdk9, cdx-1, CDX2, cdx-4, CFF, chxlO, CLIM1, CLIM2, CNBP, coS, COUP, CP1, CP1A, CP C, CP2, CPBP, CPEB binding protein, CREB-2, CRE-BP1, CRE-BPa, CREB- α, CREB-1, CREB-BPa, CRF, crx, CSBP-1, CTCF, CTF, CTF-1, CTF-2, CTF-3, CTF-5, CTF-7, CUP, CUTL1, cx, cyclin A, cyclin T1, cyclin T2a, cyclin T2b, DAP, DAX1, DB1, DBF4, DBP, dbpA, dbpAv, dbpB, DDB, DDB-1, DDB-2, DEF, δCREB, δMax, DF-1, DF-2, DF-3, dlx-1, dlx-2, dlx-3, dlx (long isoform), dlx-4 (short isoform, dlx-5, dlx-6, DP-1, DP-2, DSIF-P14, DSIF-P160, DTF, DUX1, DUX2, DUX3, DUX4, E, E12, E2F, E F+E4, E2F+p107, DUF-1, E2F-2, E2F-3, E2F-4, E2F-6, F-3, E2F-4, F-6 and 5-6E 47, E4BP4, E4F, E F1, E4TF2, EAR2, EBP-80, EC2, EF1, EF-C, EGR1, EGR2, EGR3, EIIaE-A, EIIaE-B, EIIaE-C alpha, EIIaE-C beta, eivF, EIf-1, EIk-1, emx-1, emx-2, emx-2, en-1, en-2, ENH-bind.prot, ENKTF-1, EPAS1, εF1, ER, erg-1, erg-2 ERR1, ERR2, ETF, ets-1 delta Vil, ets-2, evx-1, F2F, factor 2, factorname, FBP, F-EBP, FKBP59, FKHL18, FKHRL1P2, fli-1, fos, FOXB1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3, FOXF1, FOXF2, FOXG1a, FOXG1b, FOXG1C, FOXH1, FOXI1, FOXJ1a, FOXJ1b, FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXK1a, FOXK1b, FOXK1c, FOXL1, FOXM1a, FOXM1b, FOXM1c, FOXN1, FOXN2, FOXN3, FOXO1a, FOXO1b, FOX02, FOX03a, FOX03b, FOX04, FOXP1, FOXP3, fra-1, fra-2, FTF, FTS, G factors factor G6, GABP-alpha, GABP-beta 1, GABP-beta 2, GADD153, GAF, gamma CMT, gamma CAC1, gamma CAC2, GATA-1, GATA-2, GATA-3, GATA-4, GATA-5, GATA-6, gbx-1, gbx-2, GCF, GCMa, GCNS, GF1, GLI3, GRalpha, GRbeta, GRF-1, gsc1, GT-IC, GT-IIA, GT-IIB alpha, GT-IIB beta H1TF1, H1TF2, H2RIIBP, H4TF-1, H4TF-2, HAND1, HAND2, HB9, HDAC1, HDAC2, HDAC3, hDaxx, thermally induced factor, HEB1-p67, HEB1-p94, HEF-1B, HEF-1T, HEF-4C, HEN1, HEN2, hesx1, hex, HIF-1 alpha, HIF-1 beta, hiNF-A, hiNF-B, HINF-C, HINF-D, hiNF-D3, HIF-A, hiNF-B, HINF-C, HINF-D, hiNF-D3 HiNF-E, hiNF-P, HIP1, HIV-EP2, hlf, HLTF, HLTF (Met 123), HLX, HMBP, HMG I (Y), HMGY, HMGI-C, HNF-1A, HNF-IB, HNF-1C, HNF-3, HNF-3 alpha, HNF-3 beta, HNF-3 gamma, HNF4, HNF-4 alpha, HNF4 alpha 1, HNF-4 alpha 2, HNF-4 alpha 3, HNF-4 alpha 4, HNF4 gamma, HNF-6 alpha, hnRNPK, HOX11, HNF-4 alpha, HOXA1, HOXA10PL2, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9A, HOXA9B, HOXB-1, HOXB13, HOXB2, HOXB3, HOXB4, HOXBs, HOXB6, HOXA5, HOXB7, HOXB8, HOXB9, HOXC10, HOXC11, HOXC12, HOXC13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXD10, HOXD11 HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, hp55, hp65, HPX42B, hrpF, HSF, HSF1 (long), HSF1 (short), HSF2, hsp56, hsp90, IBP-1, ICER-II, ICER-li gamma, ICSBP, id1H', id2, id3/Heir-1, IF1, igPE-2, igPE-3, Iκ B, I κB-alpha, IκB-beta, IκBR, II-1RF IL-6RE-BP, 11-6RF, INSAF, IPF1, IRF-2, B, IRX2a, irx-3, irx-4, ISGF-1, ISGF-3 alpha, ISGF-3 gamma, lst-1, ITF-1, ITF-2, irx-3, irx-4, ISGF-3 alpha, ISGF-3 gamma, ITF-1, ITF-2, irx-3 alpha, irx-1, irx-3 alpha, irx-2, or the like JRF, jun, junB, junD, KBP-1, KER-1, kox1, KRF-1, ku autoantigen, KUP, LBP-1a, LBX1, LCR-F1, LEF-1B, LF-A1 LHX1, LHX2, LHX3a, LHX3B, LHXS, LHX6.1a, LHX6.1b, LIT-1, lmo2, LMX1A, LMX1B, L-My1 (long form), L-My1 (short form), L-My2, LSF, LXRalpha, lyF-1, lyl-1, M factor, mad1, MASH-1, max2, MAZ1, MB67, MBF1, MBF2, MBF3, MBP-1 (1), MBP-1 (2), MBP-2, MDBP, MEF-2B, MEF-2C (433 AA form), MEF-2C (465 AA form), MEF-2C (473M form), MEF-2C/delta 32 (441 AA form), MEF-2D00, MEF-2D0B, MEF-2DA0, MEF-2DAO, MEF-2DAB, MEF-2DA' B, meis-1, meis-2a, meis-2B, meis-2C, meis-2D, meis-2E, meis3, meox1a, meox2, MHox (K-2), mi, MIF-1, miz-1, MM-1, MOP3, MR, msx-1, msx-2, MTB-Zf, MTF-1, MTF 1, mxil, myb, myc, myc1, myf-3, myf-4, myf-5 Myf-6, myoD, MZF-1, NCI, NC2, NCX, NELF, NER1, net, NF Ill-a, NF NF NF-1, NF-1A, NF-1B, NF-1X, NF-4FA, NF-4FB, NF-4FC, NF-A, NF-AB, NFAT-1, NF-AT3, NF-Atc, NF-Atp, NF-Atx, nfβ A, NF-CLEOa, NF-CLEOb, NF delta E3A, NF delta E3B, NF delta E3C, NF delta E4A, NF delta E4B, NF delta E4C, nfe, NF-E, NFE, NF-E2p45, NF-E3, NFE-6, NF-Gma, NF-GMb, NF-IL-2A, NF-IL-2B, NF-jun, NF- κ B, NF- κB (sample), NF- κB1, NF-B1 precursor, NF- κB2, NF- κB2 (p 49), NF- κB2 precursor, NF- κE1, NF- κE2, NF- κE3, NF-MHCIIA, NF-MHCIIB, NF-muE, NF-muE2, NF-muE3, NF-S, NF-X, NF-X1, NF-X2, NF-X3, NFXc, NF-YA, NF-Zc, NF-Zz, NHP-1, NHP-2, NHP3, NHP-4 NHP4, NKX2-5, NKX2B, NKX2C, NKX2G, NKX3A, NKX3Av1, NKX3Av2, NKX3Av3, NKX3Av4, NKX3B, NKX A, nmi, N-Myc, N-Oct-2α, N-Oct-2β, N-Oct-3, N-Oct-4, N-Oct-5a, N-Oct-5B, NP-TCII, NR2E3, NR4A2, nrf1, nrf-1 Nrf2, NRF-2β1, NRF-2γ1, NRL, NRSF form 1, NRSF form 2, NTF, 02, OCA-B, oct-1, oct-2, oct-2.1, oct-2B, oct-2C, oct-4A, oct4B, oct-5, oct-6, octa-factor, octamer-binding factor, oct-B2, oct-B3, otx1, otx2, OZF, p107, p130, p28 modulator, p300, p38erg, p45, p49erg, p53, p55erg, p65 delta, p67, pax-1, pax-2, pax-3, A, pax-3B, pax-4, pax-5, pax-6/Pd-5a, pax-7, pax-8a, pax-8d, pax-8 and Pax-8d, pax-8e, pax-8f, pax-9, pbx-1a, pbx-1B, pbx-2, pbx-3a, pbx-3B, PC2, PC4, PC5, PEA3, PEBP2 alpha, PEBP2 beta, pit-1, PITX2, PITX3, PKNOX1, PLZF, POB, pontin52, PPARalpha, PPARgamma 1, PPARgamma 2, PPUR, PR, PRA, pRb, PRD1-BF1, PRDI-Bfc, prop-1, PSE1, P-TEFb, PTF, PTF alpha, PTFbeta, PTFdelta, PTFgamma, pubox binding factor (BJA-B), PU.1, puF, pur factor, R1, R2, RAR-alpha 1, RAR-beta 2, RAR-gamma 1, RBP60, RBP-J, rel, relA, relB, RFX, RFX, X2, RFX3, RFX-38 alpha, RFR-26 alpha, RFR-alpha, RFR 2 RORα3, RORbeta, RORgamma, rox, RPF1, RPGalpha, RREB-1, RSRFC4, RSRFC9, RVF, RXR-alpha, RXR-beta, SAP-1a, SAP-1B, SF-1, SHOX2a, SHOX2B, SHOXa, SHOXb, SHP, SIII-P110, SIII-P15, SIII-P18, SIM', six-1, six-2, six-3, six-4, six-5, six-6, SMAD-1, SMAD-2, SMAD-3, SMAD-4, SMAD-5, SOX-11, SOX-12, sox-4, sox-5, SOX-9, sp1, sp2, sp3, sp4, sph factor, spi-B, SPIN, SRCAP, SREBP-1a, SREBP-1B, SREBP-1c, SRP-2, SRE-ZBP, SRF, SRY, SRP, staf-50, STAT1, STAT2, STAT4, STAT2, STAT3 STAT6, T3R, T3R-. Alpha.1, T3R-. Alpha.2, T3R-. Beta., TAF (I) 110, TAF (I) 48, TAF (I) 63, TAF (II) 100, TAF (II) 125, TAF (II) 135, TAF (II) 170, TAF (II) 18, TAF (II) 20, TAF (II) 250. Delta., TAF (II) 28, TAF (II) 30, TAF (II) 31, TAF (II) 55, TAF (II) 70-. Alpha., TAF (II) 70-. Beta., TAF (II) 70-. Gamma., TAF-I, TAF-II, TAF-L, tal-1, tal-1. Beta., tal-2, TAR factors, TBP, TBXIA, TBXIB, TBX, TBX4, TAF (II) 28 TBXS (long isoform), TBXS (short isoform), TCF-1A, TCF-1B, TCF-1C, TCF-1D, TCF-1E, TCF-1F, TCF-1G, TCF-2α, TCF-3, TCF-4 (K), TCF-4B, TCF-4E, TCF β1, TEF-1, TEF-2, tel, TFE3 TFEB, TFIIA, TFIIA-alpha beta precursor, TFIIA-alpha/beta precursor, TFIIA-gamma, TFIIB, TFIID, TFIIE, TFIIE-alpha, TFIIE-beta, TFIIF-alpha, TFIIF-beta, TFIIH-CAK, TFIIH-cyclin H, TFIIH-ERCC2/CAK, TFIIH-MAT1, TFIIH-M015, TFIIH-P34, TFIIH-P44, TFIIH-P62, TFIIH-P80, TFIIH-P90, TFII-I, tf-LF1, tf-LF2, TGIF2, TGT3, THRA1, TIF2, TLE1, TLX3, TMF, TR2-11, TR2-9, TR3, TR4, TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREF2, TRF (2), TTF-1, TXRE BP, txREF, UBF, UBP-1 UEF-1, UEF-2, UEF-3, UEF-4, USF1, USF2b, vav, vax-2, VDR, vHNF-1A, vHNF1B, vHNF-1C, VITF, WSTF, WT1, WT1I, WT I-KTS, WT1I-del2, WT1-KTS, WT1-del2, X2BP, XBP-1, XW-V, XX, YAF2, YB-1, YEBP, YY1, ZEB, ZF1, ZF2, ZFX, ZHX1, ZIC2, ZID, ZNF174, ASH1L, ASH2 ATF2, ASXL1, BAP1, bc110, bmil, BRG1, CARM1, KAT3A/CBP, CDC73, CHD1, CHD2, CTCF, DNMT1, DOTL1, EHMT1, ESET, EZH1, EZH2, FBXL10, FRP (Plu-1), HDAC1, HDAC2, HMGA1, hnRNPA1, HP1 gamma, hset1b, jarid1A, jarid1C, KIAA1718JHDM1D, KAT5, KMT4, LSD1, NFKB P100, NSD2 MBD2, MBD3, MLL2, MLL4, P300, pRB, rbAP46/48, RBP1, rbBP5, ringing ib, RNApolII PS2, RNApolII PS5, ROC1, sap30, setDB1, sf3b1, SIRT6, SMYD1, SP1, SUV39H1, SUZ12, TCF4, TET1, TRRAP, TRX2, WDR5, H3K27me3, H3K4me3, WDR77, and/or YY1.
In one embodiment of the present invention, in one embodiment, the secondary antibody is goat anti-mouse, goat anti-rat, goat anti-rabbit, goat anti-donkey, goat anti-chicken, goat anti-human, goat anti-guinea pig, rabbit anti-mouse, rabbit anti-rat, rabbit anti-goat, rabbit anti-donkey, rabbit anti-human, rabbit anti-guinea pig, rabbit anti-chicken, mouse anti-rabbit, mouse anti-rat, mouse anti-donkey, mouse anti-chicken, mouse anti-human, mouse anti-guinea pig, mouse anti-goat, rat anti-rabbit, rat anti-mouse, rat anti-donkey, mouse anti-donkey rat-resistant chicken, rat-resistant human, rat-resistant guinea pig, mouse-resistant goat, donkey-resistant rabbit, donkey-resistant rat, donkey-resistant mouse, donkey-resistant chicken, donkey-resistant human, donkey-resistant guinea pig, donkey-resistant goat, chicken-resistant rabbit, chicken-resistant rat, chicken-resistant donkey, chicken-resistant mouse, chicken-resistant human, chicken-resistant guinea pig, chicken-resistant goat, guinea pig-resistant rabbit, guinea pig-resistant rat, guinea pig-resistant donkey, guinea pig-resistant chicken, guinea pig-resistant human, guinea pig-resistant mouse, and guinea pig-resistant goat.
Best mode (examples)
Example 1:
preparation of transposase-streptavidin
Any method known in the art may be used to generate the transposase-streptavidin fusion protein. For example, tn 5-streptavidin fusion protein can be expressed recombinantly in E.coli.
Preparation of transposase-biotin
Any method known in the art may be used to biotinylated the transposase. For example, tn5 (Vazyme, cat. S601) can be labeled with NHS-activated biotin (Invitrogen, cat. 2174564). See, e.g., muraoka et al Analytical Biochemistry,557,46-58,2018.
The biotin-modified oligonucleotides can also be synthesized using any method known in the art to form complexes with transposases (see below "transposase-biotin: avidin: biotin-secondary antibody assembly, scheme 2").
Preparation of secondary antibody-biotin
The secondary antibody may be biotinylated using any method known in the art. For example, goat anti-rabbit IgG H & L secondary antibody (Abcam, cat No. ab 6702) can be labeled with NHS activated biotin (Invitrogen, cat No. 2174564). See, e.g., muraoka et al Analytical Biochemistry,557,46-58,2018.
Transposase-avidin-biotin-secondary antibody assembly
The transposase-streptavidin fusion protein was mixed with the biotinylated secondary antibody in PBS at a molar ratio of 1:5 and incubated for 1 hour at room temperature.
Transposase-biotin-avidin-biotin-second antibody Assembly, scheme 1
Streptavidin (Invitrogen, cat. No. S888) was mixed with biotinylated secondary antibody in PBS at a molar ratio of 1:1 and incubated for 1 hour at room temperature to give the intermediate avidin, biotin-secondary antibody. The intermediate was mixed with biotinylated transposase in PBS at a molar ratio of 5:7 and incubated for 1 hour at room temperature.
Transposase-biotin-avidin-biotin-second antibody Assembly, scheme 2
The biotin modified primer is synthesized in the biological engineering company: primerA, primer B, primer C (see appendix), using PBS to dissolve PrimerA, primerB, primer C to 10. Mu.M, taking 10. Mu.l Primer A+10. Mu.l Primer B to compose reaction 1, taking 10. Mu.l Primer A+10. Mu.l Primer C to compose reaction 2, vortexing reaction 1 and reaction 2 respectively, thoroughly mixing them, and briefly centrifuging to bring the solution back to the bottom, placing it in a PCR instrument, and carrying out the following reaction procedure:
after the reaction was completed, the reaction 1 product and the reaction 2 product were mixed in equal volumes and named AdapterMix.
Subsequently, the following reaction components were added sequentially to a sterilized PCR tube:
component (A) 2 mug preparation System
Tn5(500ng/μl) 4μl
AdapterMix 7μl
PBS 39μl
The mixture was gently applied with a pipette for 20 times, and the mixture was allowed to react at 30℃for 1 hour. The reaction product is named as BT-Tn5 and is preserved at the temperature of minus 30 to minus 15 ℃.
The biotin-labeled secondary antibody, namely secondary antibody-BT, is incubated with SA protein and BT-Tn5 in a molar ratio of 5:7:7 for 1h at room temperature in PBS for later use, and is named as: secondary antibody-BT, SA, BT-Tn5 complex.
Wherein the secondary antibody-BT is SA, BT-Tn5-1 is primer A (5' -phos-CTGTCTCTTATACACAT) BT CT BT -3')+primerB(5′-TCGT BT CGGCAGCGTCAGATGTGTAT BT AAGAGACAG-3′)+primer C(5′-GTCTCGTGGGCT BT CGGAGATGTGTAT BT AAGAGACAG-3); the secondary antibody-BT SA BT-Tn5-2 is primer A (5' -phos-CT) BT GTCTCTTAT BT ACACATCT-3')+primer B(5′-TCGTCGGCAGCGTCAGATGTGT BT AT BT AAGAGACAG-3′)+primer C(5′-GTCT BT CGTGGGCT BT CGGAGATGTGTATAAGAGACAG-3′)。
Assembling pG-Tn5 secondary antibody
The secondary antibody was 1:100 diluted with 100. Mu.L digitonin Wash buffer (Dig-Wash buffer, from Northey 'ne product TD 901), 0.58. Mu.L 6.88. Mu.M pG-Tn5 (from Northey' ne product TD 901) was added and incubated for 60 minutes at room temperature with 200rpm rotation. The pre-incubated secondary antibody pG-TN5 complex was obtained.
Example 2:
in the embodiment, H3k4me3 histone modification is taken as an exemplary analysis target, and experimental comparison is carried out by using a secondary antibody-BT: SA-Tn5 complex and a conventional pG-Tn5 complex so as to illustrate the steps and beneficial effects of the method.
The library construction reagents In this example were all from the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pG-Tn 5) (cat# TD 901) kit of Nanjinouzan biotechnology Co., ltd, and the specific experimental procedures were as follows:
buffer preparation and ConA bead pretreatment were performed according to the seventh and ninth sections of the TD901 kit instructions. The secondary antibody-BT: SA-Tn5 complex was constructed as described in example 1.
1. Cell collection
The same 6 samples of HeLa cells were taken, 100. Mu.l each (about 100000 HeLa cells), centrifuged at 2500rpm for 3min at room temperature and the supernatant discarded. To each sample was added 500. Mu.l of Wash Buffer (Wash Buffer) at room temperature to resuspend the cells, centrifuged at 2500rpm for 3min and the supernatant discarded for subsequent experiments.
Wherein, the first Hela cell sample uses Anti-Histone H3 (mono methyl K4) Anti (abcam company, cat# ab 8895) as primary antibody and pG-Tn5 complex for the first reference library construction (sample 1); the second Hela cell sample was subjected to a first negative control library construction using Rabbit (DA 1E) mAb IgG (Cell Signaling Technology, cat# 66362S) as primary antibody and pG-Tn5 complex (sample 2).
A third Hela cell sample was subjected to a second reference library construction using Anti-Histone H3 (mono methyl K4) Anti as primary and secondary antibodies, pG-Tn5 complex (sample 3); a fourth sample of Hela cells was subjected to a second negative control library construction using Rabbit (DA 1E) mAb IgG as primary and secondary antibodies pG-Tn5 complex (sample 4).
The fifth Hela cell sample was tested for library construction using Anti-Histone H3 (mono methyl K4) Anti as primary and secondary antibodies-BT: SA-Tn5 complex (sample 5); a sixth sample of Hela cells was subjected to a third negative control library construction using Rabbit (DA 1E) mAb IgG as primary and secondary antibodies-BT: SA-Tn5 complex (sample 6).
2. Incubation of cells with ConA beads
4 cell samples were incubated with ConA beads, respectively, according to the ninth section of the TD901 product instruction.
3. Incubation with primary antibody
Mu.l of pre-chilled Antibody Buffer (anti-body Buffer) was added to the individual samples, the cells were resuspended and gently vortexed to mix and placed on ice.
To the EP tube of the first, third and fifth samples, 1. Mu.l of Anti-Histone H3 (mono methyl K4) Anti-body was added, mixed by gentle vortexing, and incubated for 2H at room temperature with spin.
Mu.l of Rabbit (DA 1E) mAb IgG was added to the EP tube of the second, fourth and sixth samples, gently vortexed, and incubated for 2h at room temperature with spin.
4. Incubation of secondary antibodies and/or Tn5
Six samples after the primary antibody incubation were subjected to secondary antibody incubation according to the ninth section of the TD901 product instructions.
Wherein the first and second samples are incubated with goat anti-rabbit IgG H & L secondary antibody; incubating the third sample and the fourth sample with a secondary antibody pG-Tn5 complex; the fifth and sixth samples were incubated with the secondary antibody-BT: SA-Tn5 complex.
The first and second samples after the second antibody incubation need to be incubated with pG-Tn5, and the operations are performed according to the ninth part of the TD901 product specification.
Because the complex in the incubation of the fifth and sixth sample secondary antibodies contains Tn5 transposomes, compared with the conventional pG-Tn5 complex library building mode, the secondary antibody-BT: SA-Tn5 complex library building mode shortens the incubation time of the transposomes by 1 hour, and the secondary antibody-BT: SA-Tn5 complex library building mode does not need extra secondary antibodies to be rinsed, so that the overall experiment time is shortened by 1 hour.
5. Fragmentation reaction
The fifth and sixth samples incubated with the secondary antibody-BT: SA-Tn5 complex, the first and second samples incubated with pG-Tn5, and the third and fourth samples incubated with the secondary antibody-pG-Tn 5 complex were subjected to fragmentation reaction, and incubated at 37℃for 1 hour.
6. DNA extraction and purification
According to the ninth part of the TD901 product specification, DNA extraction and purification are carried out on the fragmented first and second samples; the third, fourth, fifth and sixth samples after fragmentation were directly added with 30. Mu.l of 1 XTE without DNA extraction and purification, and the samples were directly subjected to PCR amplification reaction.
The fifth and sixth samples do not require DNA extraction and purification, and therefore are shortened in time by approximately 3 hours relative to the first and second samples that require extraction and purification.
7. Library amplification
Library amplification was performed according to the ninth section of the TD901 product specification, wherein the P5/P7 primer was used with the Nanjinozan Biotechnology Co., ltd. TruePrep Index KitV for Illumina kit (cat. No. TD 202).
8. PCR product purification
The PCR amplified product was subjected to magnetic bead purification according to the ninth section of the TD901 product specification, wherein the magnetic bead used was the Nanjenofizan Biotechnology Co., ltd VAHTS DNA CleanBeads kit (cat. No. N411).
9. Library quality detection
The prepared library is subjected to library length distribution detection on an Agilent 2100Bioanalyzer (FIG. 6), and the obvious ladder-shaped library distribution of pG-Tn5 group (namely sample 1) and secondary antibody-BT: SA-Tn5 group (namely sample 5) can be found, so that the secondary antibody-BT: SA-Tn5 technical scheme can be normally built, but the ladder peak of the secondary antibody: PG-Tn5 group (namely sample 3) is not obvious, so that the cleavage efficiency of Tn5 enzyme on chromatin can be influenced.
13. Second generation sequencing
The library was diluted to 5ng/ul and 15 μl was sent to Nanjing age and Gene Biotechnology Inc. for sequencing with HiseqX as a sequencing instrument.
Sequencing results
The data shows that in the negative control IgG experimental group, compared with pG-Tn5 experimental scheme (sample 2) and secondary antibody PG-Tn5 experimental scheme (sample 4), the secondary antibody BT SA-Tn5 experimental scheme (sample 6) has the IgG signals randomly distributed, so that the false positive signals of the TSS region are reduced (figure 4); IGV view shows that the secondary antibody-BT: SA-Tn5 group (sample 5) positive signal is more enriched and the IgG control (sample 6) false positive signal is reduced (FIG. 5); library peaks show that the secondary antibodies-BT: SA-Tn5 group (sample 5) had a distribution of nucleosome ladders compared with pG-Tn5 group (sample 1), while the secondary antibodies-PG-Tn 5 group (sample 3) had no apparent distribution of nucleosome ladders, suggesting that they may affect the cleavage efficiency of Tn5 enzyme on chromatin (FIG. 6).
Similar experimental results were obtained for CTCF transcription factors, H3K27me3 and H3K4me3 histone modifications as targets.
Example 3:
in the embodiment, H3k4me3 histone modification is taken as an exemplary analysis target, and experimental comparison is carried out by using a secondary antibody-BT: SA: BT-Tn5-1 complex, a secondary antibody-BT: SA: BT-Tn5-2 complex, a secondary antibody-BT: SA-Tn5 complex and a conventional pG-Tn5 complex so as to illustrate the steps and beneficial effects of the method.
Wherein the procedure of experimental group of "secondary antibody-BT: SA-Tn5 complex" and conventional "pG-Tn5 complex" is the same as in example 2; the experimental procedures of "secondary antibody-BT: SA-Tn 5-1 complex" and "secondary antibody-BT: SA-BT-Tn 5-2 complex" were identical to those of the "secondary antibody-BT: SA-Tn5 complex" set in example 2, except that "secondary antibody-BT: SA-Tn5 complex" was replaced with "secondary antibody-BT: SA-Tn 5-1 complex" or "secondary antibody-BT: SA-Tn 5-2 complex".
Sequencing results
The on-press data shows that in the negative control IgG experimental group, the IgG of the "secondary antibody-BT: SA: BT-Tn5-1 complex" and the "secondary antibody-BT: SA: BT-Tn5-2 complex" have no obvious TSS enrichment (FIG. 7); IGV views show that, compared with the conventional pG-Tn5 complex (primary antibody+secondary antibody+pGTn 5), the primary antibody+secondary antibody-BT: SA: BT-Tn5-1 complex, the primary antibody+secondary antibody-BT: SA: BT-Tn5-2 complex and the primary antibody+secondary antibody-BT: SA-Tn5 can effectively reduce the IgG background and improve the specific signal of the antibody (figure 8); library peak pattern shows that the library has obvious distribution of nucleosome ladder, which indicates that the complexes constructed by different modification modes can be successfully built into the library (figure 9).
Similar experimental results were obtained for CTCF transcription factors, H3K27me3 and H3K4me3 histone modifications as targets.
Appendix: transposome oligonucleotide sequences (wherein bold, italic, underlined bases are biotin-modified bases)
PrimerA comprises the sequence of SEQ ID NO: 1. SEQ ID NO:4 to SEQ ID NO:182
PrimerB comprises the sequence of SEQ ID NO: 2. SEQ ID NO:183 to SEQ ID NO:284Primer C comprises SEQ ID NO: 3. SEQ ID NO:285 to SEQ ID NO:464SEQ ID NO:1
CTGTCTCTTATACACATCT
SEQ ID NO：2
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
SEQ ID NO：3
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
SEQ ID NO：4
SEQ ID NO：5
SEQ ID NO：6
SEQ ID NO：7
SEQ ID NO：8
SEQ ID NO：9
SEQ ID NO：10
SEQ ID NO：11
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/>
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/>
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/>
SEQ ID NO：100
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/>
SEQ ID NO：133
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/>
SEQ ID NO：166
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/>
SEQ ID NO：199
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/>
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/>
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/>
SEQ ID NO：298
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/>
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/>
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/>
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/>
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/>

Claims (10)

1. A biotin-avidin-based secondary antibody transposase complex represented by the formula:
transposase-avidin (biotin-second antibody) n Wherein n is 1, 2, 3 or 4.
2. The complex of claim 1, wherein the avidin is streptavidin.
3. The complex of claim 1, wherein the transposase is a Tn5 transposase.
4. A mixture of complexes according to any one of claims 1 to 3, represented by the formula:
transposase-avidin (biotin-second antibody) p Wherein p is greater than 1 and less than 4.
5. A biotin-avidin-based secondary antibody transposase complex represented by the formula:
(transposase-biotin) m Avidin (biotin-second antibody) n Wherein m and n are each independently 1, 2 or 3, and m+n is less than or equal to 4.
6. The complex of claim 5, wherein the avidin is streptavidin.
7. The complex of claim 5, wherein the transposase is a Tn5 transposase.
8. The complex of claim 5, wherein the biotin is attached to an oligonucleotide of the transposome, optionally the oligonucleotide is a partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3 '(SEQ ID NO: 12) and 5' -TCGT BT CGGCAGCGTCAGATGTGTAT BT AAGAGACAG-3 '(SEQ ID NO: 195) and/or partially paired 5' -phos-CTGTCTCTTATACACAT BT CT BT -3 '(SEQ ID NO: 12) and 5' -GTCTCGTGGGCT BT CGGAGATGTGTAT BT AAGAGACAG-3 (SEQ ID NO: 297) or alternatively the oligonucleotide is a partially paired 5' -phos-CT BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -TCGTCGGCAGCGTCAGATGTGT BT AT BT AAGAGACAG-3 '(SEQ ID NO: 215) and/or partially paired 5' -phos-CT BT GTCTCTTAT BT ACACACATCT-3 '(SEQ ID NO: 29) and 5' -GTCT BT CGTGGGCT BT CGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：317)，T BT Representing biotinylated T.
9. A mixture of complexes according to any one of claims 5 to 8, represented by the formula:
(transposase-biotin) q Avidin (biotin-second antibody) p Wherein p and q are each greater than or equal to 1 and less than or equal to 3, and q+p is less than or equal to 4.
10. A method of detecting interaction of a cell with DNA at a target protein, comprising:
(1) Incubating the cells with a primary antibody directed against the target protein;
(2) Incubating a cell with a complex of any one of claims 1-3 and 5-8 or a mixture of claim 4 or 9, wherein the secondary antibody in the complex is a secondary antibody specific for the primary antibody and allowing transposase to act on the genome of the cell;
(3) Amplifying the cellular DNA;
(4) Purifying the amplified DNA; and is combined with
(5) The amplified DNA was sequenced.
CN202310316670.3A 2022-04-06 2023-03-29 Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA Pending CN116891532A (en)

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CN112795563A (en) * 2021-03-23 2021-05-14 上海欣百诺生物科技有限公司 Use and method of biotinylated transposomes for recovering CUT & Tag or ATAC-seq products
CN113322254A (en) * 2021-01-06 2021-08-31 南京诺唯赞生物科技股份有限公司 Methods and tools for studying multi-target protein-DNA interactions

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US20180245069A1 (en) * 2017-02-21 2018-08-30 Illumina, Inc. Tagmentation using immobilized transposomes with linkers
US20210139985A1 (en) * 2018-04-10 2021-05-13 Board Of Regents, The University Of Texas System Dna-barcoded antigen multimers and methods of use thereof
CN113322254A (en) * 2021-01-06 2021-08-31 南京诺唯赞生物科技股份有限公司 Methods and tools for studying multi-target protein-DNA interactions
CN112553695A (en) * 2021-02-23 2021-03-26 翌圣生物科技（上海）有限公司 Rapid library construction method for identifying target protein chromatin binding map
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