Method for simultaneously enriching disordered protein and combining three-dimensional genome structure

Abstract

The invention provides a method for simultaneously enriching disordered protein and a combined three-dimensional genome structure thereof, and relates to the technical field of molecular biology. The method comprises the following steps: after cross-linking cells with formaldehyde and disuccinimide glutaric acid (DSG), carrying out enzyme digestion treatment, terminal repair and adjacent connection by using restriction enzyme Dpn II; after connection, ultrasonically breaking the cell nucleus to obtain an ultrasonically broken product, dividing the ultrasonically broken product into two parts, capturing DNA and a protein complex of one part of the product by using biotinylated isoxazole, digesting protein by using protease, extracting and purifying the DNA, and carrying out library building and sequencing on the purified DNA; the other part of the product is treated by nuclease, protein is extracted, and then protein spectrum analysis is carried out. The method can realize whole genome non-destructive detection of the disordered protein and the three-dimensional structure of the chromatin combined with the disordered protein, and provides an effective way for structural analysis of the disordered protein.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for simultaneously enriching disordered proteins and combining them with a three-dimensional genome structure. Background Art

[0002] Intrinsically disordered proteins (IDPs), which account for 1 / 3 of the human proteome, are characterized by the lack of a stable three-dimensional structure under physiological conditions and play an important role in various biological processes. IDPs have the ability to form biomolecular condensates through phase separation, a process that promotes the reorganization of three-dimensional chromatin structure, thus having a significant impact on gene regulation, development, and disease progression. For example, heterochromatin protein 1 (HP1) mediates phase separation, leading to chromatin compaction and the formation of heterochromatin domains. Similarly, CTCF enhances chromatin AA compartment interactions, maintains the self-renewal of embryonic stem cells (ESCs), and inhibits their differentiation into neural progenitor cells. In addition, UTX is an important tumor suppressor with a strong ability to phase separate. When its intrinsically disordered regions (IDRs) are missing, UTX loses its ability to condense, leading to tumorigenesis.

[0003] Currently, there are two main strategies to study the relationship between IDPs and the three-dimensional structure of chromatin. The Casl-Drop system allows the induction of liquid condensates by controlling IDPs at specific genomic sites, thereby enabling the observation of changes in the three-dimensional structure of chromatin. Another method can destroy the characteristics of intracellular phase separation by combining 1,6-hexanediol, and detect the effects of phase separation on changes in the three-dimensional structure of chromatin by treating it for different periods of time. However, this method cannot understand the changes in disordered proteins and the three-dimensional structure of chromatin in the original state of the cell. Therefore, there is still a lack of methods that can achieve non-destructive, genome-wide detection of disordered proteins and the three-dimensional structure of chromatin.

[0004] Biotinylated isoxazole (b-isox) is a small molecule that can enrich a wide range of disordered proteins by forming microcrystals. Its discovery has become a powerful tool for exploring IDP proteomics. Recent studies have used b-isox to systematically identify potential phase-separated proteins in Arabidopsis and rice. In addition, in 2023, the DisP-seq method used b-isox precipitation combined with next-generation sequencing to provide new insights into protein-DNA interactions. This method identified the one-dimensional features of the genome bound by disordered proteins, but the technology failed to identify the three-dimensional structural features of disordered proteins and chromatin. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for simultaneously enriching disordered proteins and their binding to three-dimensional genome structures, which can simultaneously enrich disordered proteins and their mediated and / or binding three-dimensional structures.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a method for simultaneously enriching disordered proteins and combining them with three-dimensional genome structures, comprising the following steps: cross-linking and lysing cells to be tested to extract and obtain cell nuclei; using restriction endonuclease Dpn II to perform enzyme digestion, end repair and proximity ligation on the obtained cell nuclei; ultrasonically crushing the cell nuclei after proximity ligation to obtain ultrasonic crushing products; dividing the ultrasonic crushing products into two to obtain product I and product II, enriching the complex of DNA and protein after ultrasonic crushing with biotinylated isoxazole in product I, washing, eluting and protease treating the complex of DNA and protein; extracting and purifying DNA after protease treatment, and sequencing the purified DNA; treating product II with nuclease, extracting protein, and then performing protein spectrum analysis.

[0007] Preferably, the cells to be tested are cross-linked using formaldehyde and disuccinimidyl glutarate.

[0008] More preferably, the cross-linking comprises the following steps: resuspending the cells to be tested in a 1×PBS solution, adding formaldehyde and incubating for 8 to 15 minutes, and then adding glycine to terminate the reaction; washing with a 1×PBS solution after terminating the reaction, resuspending with a 1×PBS solution containing 3 mM disuccinimidyl glutaric acid, incubating at room temperature for 30 to 50 minutes, and then adding glycine to continue incubation; washing with a 1×PBS solution containing 0.05% bovine serum albumin, adjusting the cell concentration, centrifuging, removing the supernatant, and collecting the cells for cryopreservation in liquid nitrogen.

[0009] Preferably, the lysis comprises the following steps: resuspending the cross-linked cells in 1× lysis buffer, adding protease inhibitors, incubating on ice for 10-20 min, centrifuging, and discarding the supernatant to obtain precipitate 1; washing precipitate 1 with 1×NEB buffer 3.1, centrifuging, and discarding the supernatant to obtain precipitate 2; resuspending precipitate 2 with 1×NEB buffer 3.1, adding 1% SDS and incubating for 8-12 min, and terminating the reaction with 10% Triton X-100.

[0010] Preferably, the end repair reaction solution includes 5-10 μL 10×NEB buffer 3.1, 1-2 μL 10 mM dCTP, 1-2 μL 10 mM dGTP, 1-2 μL 10 mM dTTP, 10-20 μL 1 mM biotin-14-dATP and 8-15 μL 5 U / μL DNA polymerase I large fragment Klenow.

[0011] Preferably, the proximity ligation reaction solution comprises 100-150 μL 10×T4 ligation buffer, 100-150 μL 10% Triton X-100, 40-60 μL T4 DNA ligase and 300-400 μL Milli-Q water.

[0012] Preferably, the ultrasonically disrupted reaction solution comprises an ultrasonic buffer containing 8-15 μL of protease inhibitors; the ultrasonic buffer comprises 40-60 mM HEPES, 120-160 mM NaCl, 0.5-2 mM EDTA, 0.5-2% Triton X-100 and 0.05-0.2% SDS.

[0013] Preferably, the method for enriching biotinylated isoxazole comprises the following steps: adding 80-120 μM biotinylated isoxazole after ultrasonic treatment, and incubating with rotation at 4° C. for 0.5-2 h.

[0014] Preferably, the washing buffer comprises 1× protease inhibitor, 0.05-0.2 mM PMSF, 10-30 mM β-mercaptoethanol, 10-30 mM Tris-HCl, 120-180 mM NaCl, 3-8 mM MgCl2, 0.1-0.8% NP-40 and 5-15% glycerol.

[0015] Preferably, the elution buffer comprises 5-15 mM Tris-HCl, 0.05-0.2% SDS, 120-180 mM NaCl and 3-8 mM DTT.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a method for simultaneously enriching disordered proteins and their binding to three-dimensional genome structures (DisP-HiC, Disordered protein-High-throughput chromosome conformation capture). The present invention uses formaldehyde and disuccinimidyl glutarate (DSG) to cross-link cells and fix the three-dimensional structural characteristics of chromatin; and further optimizes the connection system and enzyme cutting system to improve the connection efficiency; the ultrasonic fragmentation product is collected after the connection product is ultrasonically fragmented, and the product is divided into two, one part of the product uses biotinylated isoxazole to capture DNA and protein complexes, and the protein is digested by protease, and finally the DNA is sequenced by library construction; the other part of the product is treated with nuclease, protein is extracted, and protein spectrum analysis is performed. The present invention can simultaneously enrich disordered proteins and their mediated and / or bound three-dimensional structures, realize the non-destructive detection of the three-dimensional structure of chromatin bound by disordered proteins in the whole genome, and provide an effective way to study the three-dimensional structural characteristics of chromatin mediated by disordered proteins in the whole genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the experimental process of the DisP-HiC method of the present invention; Figure 2 Gel electrophoresis detection of the Dpn II digestion product and the proximity ligation product of the DisP-HiC method of the present invention; Figure 3 The ultrasonic time for obtaining the highest DNA concentration is detected after the cell nucleus is broken by different ultrasonic times in the DisP-HiC method of the present invention; Figure 4 The data analysis results of the DisP-HiC method of the present invention, in which A is the statistical data of the ratio of sequence read pairs within / between chromosomes and the ratio of interactions greater than / less than 10,000 base pairs in the DisP-HiC data, B is the calculation of the correlation of the results of three repeated experiments, and C is a heat map based on the interactions within different chromosomes / between chromatin, 1, 2, 3...X represent chromosome numbers respectively; Figure 5 The DisP-HiC method of the present invention uses biotinylated isoxazole to enrich the K562 cell proteome; Figure 6 This is a diagram showing the results of the micrococcal nuclease digestion experiment. In the figure, A is the electrophoresis of the cell nucleus digested with different enzyme dosages for 1 minute, and B is the result of proximity ligation after digestion. Figure 7This is a data quality inspection diagram after library construction and sequencing based on micrococcal nuclease digestion. A is the statistical alignment / not necessarily to the genome sequence and the repeated / non-repeated alignment sequence read pairs. B is the ratio of inter-chromosomal / intra-chromosomal sequences and insertion fragments of different sizes. C is a heat map based on the interactions within different chromosomes / chromatin after micrococcal nuclease digestion. In the figure, 1, 2, 3...X represent chromosome numbers respectively. Figure 8 To optimize the proximity ligation process based on the micrococcal nuclease digestion experiment, Figure A is the electrophoresis of cell nuclei digested with different enzyme dosages for 1 minute, and Figure B is the electrophoresis of the proximity ligation product after the digestion product was treated with sodium dodecyl sulfate. DETAILED DESCRIPTION

[0018] The invention provides a method for simultaneously enriching disordered proteins and combining them with three-dimensional genome structures, comprising the following steps: cross-linking and lysing cells to be tested to extract and obtain cell nuclei; using restriction endonuclease Dpn II to perform enzyme digestion, end repair and proximity ligation on the obtained cell nuclei; ultrasonically crushing the cell nuclei after proximity ligation to obtain ultrasonic crushing products; dividing the ultrasonic crushing products into two to obtain product I and product II, enriching the complex of DNA and protein after ultrasonic crushing with biotinylated isoxazole in product I, washing, eluting and protease treating the complex of DNA and protein; extracting and purifying DNA after protease treatment, and sequencing the purified DNA; treating product II with nuclease, extracting protein, and then performing protein spectrum analysis.

[0019] In the present invention, formaldehyde and disuccinimidyl glutaric acid are preferably used to crosslink the cells to be tested. The specific steps are preferably as follows: resuspend the cells to be tested in 1×PBS solution, add formaldehyde and incubate for 8~15min, then add glycine to terminate the reaction; after terminating the reaction, wash with 1×PBS solution, resuspend with 1×PBS solution containing 3mM disuccinimidyl glutaric acid, incubate at room temperature for 30~50min, and add glycine to continue incubation; wash with 1×PBS solution containing 0.05% bovine serum albumin, adjust the cell concentration, centrifuge, remove the supernatant, collect the cells and freeze them in liquid nitrogen. In the present invention, the volume concentration of the formaldehyde is preferably 35~40%, more preferably 37%. The final concentration of the formaldehyde is preferably 0.5~2%, more preferably 1%. The purpose of adding formaldehyde in the present invention is to fix the cells. After adding formaldehyde, it is preferably incubated at room temperature for 8~15min, and more preferably incubated at room temperature for 10min. In the present invention, glycine is used to terminate the formaldehyde reaction, and the concentration of the glycine is preferably 2-3 M, more preferably 2.5 M. The final concentration of the glycine is preferably 150-180 mM, more preferably 168 mM. In the present invention, the conditions for terminating the reaction are preferably: incubating at room temperature for 3-8 min after adding glycine, and incubating on ice for an additional 10-20 min.

[0020] In the present invention, the cells are lysed after cross-linking, and the lysis preferably includes the following steps: resuspending the cross-linked cells in 1× lysis buffer, adding protease inhibitors, incubating on ice for 10~20min, centrifuging, discarding the supernatant to obtain precipitate 1; washing precipitate 1 with 1×NEB buffer 3.1, centrifuging, discarding the supernatant to obtain precipitate 2; resuspending precipitate 2 with 1×NEB buffer 3.1, adding 1% SDS and incubating for 8~12min, and terminating the reaction with 10% Triton X-100. In the present invention, the lysis buffer preferably includes 10mM Tris-HCl, pH 8.0, 10mM NaCl, and 0.2% Igepal CA-630. The present invention preferably uses sodium dodecyl sulfate (SDS) treatment to make the enzyme cleavage more complete and improve the connection efficiency.

[0021] In the present invention, the cell nucleus is extracted after cell lysis, and the cell nucleus is subjected to enzyme digestion, end repair and proximity ligation treatment. The specific steps are preferably as follows: after cell lysis, 10-15 μL 10×NEB buffer 3.1 and 400U restriction endonuclease Dpn II are preferably added, gently mixed, and digested overnight on a shaker at 37°C; incubated at 60-68°C for 15-25 minutes to inactivate Dpn II, and then cooled on ice for end repair and proximity ligation treatment. In the present invention, the end repair reaction solution preferably includes 5-10 μL 10×NEB buffer 3.1, 1-2 μL 10mM dCTP, 1-2 μL 10mM dGTP, 1-2 μL 10mM dTTP, 10-20 μL 1mM biotin-14-dATP and 8-15 μL 5U / μL DNA polymerase I large fragment Klenow; more preferably includes 6 μL 10×NEB buffer 3.1, 1.5 μL 10mM dCTP, 1.5 μL 10mM dGTP, 1.5 μL 10mM dTTP, 15 μL 1mM biotin-14-dATP and 10 μL 5U / μL DNA polymerase I large fragment Klenow. The proximity ligation reaction solution preferably includes 100-150 μL 10×T4 ligation buffer, 100-150 μL 10% Triton X-100, 40-60 μL T4 DNA ligase and 300-400 μL Milli-Q water; more preferably includes 120 μL 10× ligation buffer, 120 μL 10% TritonX-100, 50 μL T4 DNA ligase and 375 μL Milli-Q water.

[0022] In the present invention, the cell nucleus is subjected to enzymatic cleavage, end repair and proximity ligation treatment and then ultrasonically disrupted. The specific steps of the ultrasonic disruption are preferably as follows: washing twice with an ultrasonic buffer containing 8-15 μL of protease inhibitor (Thermo Fisher, 78438), discarding the supernatant to obtain a precipitate; resuspending the precipitate in an ultrasonic buffer and transferring it to a microTUBE-130, and using a Covaris M220 ultrasonic processor for ultrasonic disruption to shear the chromatin. In the present invention, the parameter settings of the Covaris M220 ultrasonic processor are preferably 75W, 5% duty cycle, 200 pulses / burst, 12min. In the present invention, after ultrasonic disruption treatment, it is preferably centrifuged at 12000g for 8-15min to collect the supernatant; the supernatant is fixed to volume using an ultrasonic buffer.

[0023] In the present invention, an ultrasonic product is obtained after ultrasonic treatment; the ultrasonic product is divided into two to obtain product I and product II, the product I is enriched with biotinylated isoxazole to obtain a complex of DNA and protein after ultrasonic treatment, and the complex of DNA and protein is washed, eluted and treated with protease; after protease treatment, DNA is extracted and purified, and the purified DNA is sequenced for library construction, and the specific steps are preferably as follows: biotinylated isoxazole (b-isox) is added to the above-mentioned fixed volume supernatant to obtain a mixture, the mixture is rotated and incubated at 4°C for 0.5-2h, centrifuged at 13000g for 10-20min, the supernatant is removed and the precipitate is collected; the precipitate is washed twice with a washing buffer, and then centrifuged at 4°C and 13000g for 10-20min, the supernatant is removed and the precipitate is collected; the precipitate is resuspended in an elution buffer, RNaseA is added, and the mixture is shaken and incubated at 37°C and 600rpm for 25-35min. Then, proteinase K was added and shaken at 600 rpm overnight at 60-70° C. In the present invention, the washing buffer preferably includes 1× protease inhibitor, 0.05-0.2 mM PMSF, 10-30 mM β-mercaptoethanol, 10-30 mM Tris-HCl, 120-180 mM NaCl, 3-8 mM MgCl2, 0.1-0.8% NP-40 and 5-15% glycerol, more preferably includes 1× protease inhibitor, 0.1 mM PMSF, 20 mM β-mercaptoethanol, 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40 and 10% glycerol. The elution buffer preferably includes 5-15 mM Tris-HCl, 0.05-0.2% SDS, 120-180 mM NaCl and 3-8 mM DTT, and more preferably includes 10 mM Tris-HCl, 0.1% SDS, 150 mM NaCl and 5 mM DTT. The reagents involved in the present invention can be purchased through conventional channels.

[0024] In the present invention, after the disordered protein is enriched with biotinylated isoxazole, DNA is extracted and purified, and the purified DNA is subjected to library construction and sequencing. The specific steps are preferably as follows: DNA is extracted using 2×VAHTS DNA Clean Beads (N411-02, Vazyme) and eluted with Milli-Q water according to the manufacturer's instructions; in order to remove biotin from the unattached DNA ends, the reaction mixture (5 μg DNA, 5 μL 10×NEB buffer 2.1, 0.125 μL 10 mM dATP and 0.125 μL 10 mM dGTP, 5 μL T4 DNA polymerase) is incubated at 18~25°C for 3~4 minutes; DNA is purified again using VAHTS DNA Clean Beads; finally, a sequencing library is prepared for the Illumina V4 platform using a VAHTS Universal DNA Library Preparation Kit (ND610-01, Vazyme), and sequencing is performed on the Illumina platform using the TruSeq technology.

[0025] In the present invention, the product II is treated with nuclease, protein is extracted, and then protein spectrum analysis is performed. The specific steps are preferably as follows: add benzonase nuclease to product II, incubate at 35-37°C for 25-35min, remove nucleic acids in the system, centrifuge at 13000g for 8-15min, and collect the supernatant. The supernatant is subjected to protein spectrum detection. In the present invention, the benzonase nuclease is purchased through conventional channels, and the benzonase nuclease of the present invention is purchased from Merck Millipore, with a catalog number of 70664. The present invention preferably sends the supernatant to be detected by protein spectrum to BGI Protein Spectrum for detection and analysis.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0028] Example 1 A method for simultaneously enriching disordered proteins and binding to three-dimensional genome structures, comprising the following steps: 1. Cell culture 1.1. K562 human chronic myeloid leukemia cell line (ATCC, CRL-3344) was cultured in RPMI-1640 complete medium (Thermo Fisher, 31870082), supplemented with 10% fetal bovine serum (Seradigm PremiumGrade HI FBS, VWR), 2 mM l-glutamine (Thermo Fisher, 25030081) and 1× penicillin-streptomycin (Gibco, Life Technologies) in a 5% CO2, 37°C incubator. For subculture, 3.5×10 5 The cells were inoculated into 10 mL of complete culture medium and placed in a 10 cm culture dish.

[0029] 2. Cell cross-linking 2.1. The complete culture medium containing K562 human chronic myeloid leukemia cells was centrifuged at 800g for 5 min, and the cultured K562 cells were collected and completely resuspended and washed once with room temperature 1× PBS; 2.2. The cells were resuspended in 1× PBS at a concentration of 1×10 6 cells / mL; 2.3. To fix the cells, add 37% formaldehyde to a final concentration of 1% and incubate with gentle mixing for 10 min at room temperature; 2.4. Add 2.5 M glycine to a final concentration of 168 mM to terminate the formaldehyde reaction, followed by incubation at room temperature for 5 min and an additional 15 min on ice; 2.5. Centrifuge at 800g for 5 min and wash twice with room temperature 1× PBS; 2.6. Resuspend in room temperature 1× PBS containing 3 mM disuccinimidyl glutarate (DSG) and incubate at room temperature for 45 min; 2.7. Add 2.5 M glycine to a final concentration of 0.4 M, then incubate at room temperature for 5 min and on ice for an additional 15 min; 2.8. Wash twice with 4°C 1× PBS containing 0.05% bovine serum albumin (BSA); 2.9. Use 4℃ 1×PBS + 0.05% BSA to adjust the concentration to 1×10 7 cells / mL, and then the cells were dispensed into 1.5 mL low-adsorption Eppendorf EP tubes, centrifuged at 2500 g for 5 min, and the supernatant was removed; 2.10. Cells were quickly frozen in liquid nitrogen and stored at -80°C.

[0030] 3. Cell lysis and chromatin digestion 3.1. The approximately 1×10 7 The cells were resuspended in 1 mL of 4°C 1× lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630), and 10 μL of protease inhibitor (ThermoFisher, 78444) was added. After incubation on ice for 15 min, the cells were centrifuged at 2500 g for 5 min, and the supernatant was discarded. 3.2. Wash twice with 500 μL 4°C 1× NEB buffer 3.1, centrifuge at 2500 g for 5 min, and discard the supernatant; 3.3. Resuspend the pellet with 342 μL 1× NEB buffer 3.1, add 38 μL 1% SDS, incubate at 65°C for 10 min, and terminate the reaction with 43 μL 10% Triton X-100; 3.4. Add 12 μL 10×NEB buffer 3.1 and 400 U Dpn II, mix gently, and digest overnight on a shaker at 37°C.

[0031] 4. End repair and proximity ligation; 4.1. Incubate the digested product obtained in step 3 at 65°C for 20 min to inactivate Dpn II, and then cool on ice; 4.2. Prepare the end repair reaction solution, which includes 6 μL 10×NEB buffer 3.1, 1.5 μL 10 mM dCTP, 1.5 μL 10 mM dGTP, 1.5 μL 10 mM dTTP, 15 μL 1 mM biotin-14-dATP and 10 μL 5 U / μL DNA polymerase I large fragment Klenow; 4.3. Add 60 μL of end repair reaction solution to the digested nuclei and incubate at 23°C for 4 h. After cooling, add proximity ligation reaction solution (120 μL 10×T4 ligation buffer, 120 μL 10% Triton X-100, 50 μL T4 DNA ligase, 375 μL Milli-Q water), mix, and incubate at 16°C for 4 h; 5. Nuclei Sonication and Biotinylated Isoxiazole Enrichment 5.1. Centrifuge the product after proximity ligation in step 4 at 2800 g for 10 min, discard the supernatant, wash twice with 1 mL of ultrasonic buffer (50 mM HEPES, pH 7.8; 140 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% SDS) containing 10 μL of protease inhibitor (Thermo Fisher, 78438), and discard the supernatant to obtain the precipitate; 5.2. Resuspend the pellet in 130 μL of sonication buffer and transfer to microTUBE-130; 5.3 Chromatin shearing was performed using a Covaris M220 ultrasonic processor (75 W, 5% duty cycle, 200 pulses / burst, 12 min); 5.4.1 Centrifuge at 2000g for 10 min and collect the supernatant; adjust the supernatant to 600 μL using ultrasonic buffer; 5.5. The 600 μL supernatant obtained in step 5.4 was equally divided into two portions to obtain product I and product II; 100 μM biotinylated isoxazole was added to product I, and the mixture was incubated with rotation at 4°C for 1 h; 5.6. Centrifuge at 4°C, 13000g for 15 min, remove the supernatant and collect the precipitate, wash the precipitate twice with washing buffer, centrifuge at 4°C, 13000g for 15 min, remove the supernatant and collect the precipitate; the washing buffer comprises 20mM Tris-HCl, pH 7.4; 150mM NaCl; 5mM MgCl2; 0.5% NP-40; 10% glycerol, plus 1× protease inhibitor, 0.1mM PMSF and 20mM β-mercaptoethanol; 5.7. Resuspend the precipitate collected in step 5.6 in 200 μL elution buffer (10 mM Tris-HCl, pH 8.0; 0.1% SDS; 150 mM NaCl; 5 mM DTT), add 1 μL RNase A and incubate at 37°C with shaking at 600 rpm for 30 min. Then, add 10 μL proteinase K and shake at 65°C with shaking at 600 rpm overnight.

[0032] 6. DNA purification and library preparation 6.1. Extract DNA using 2×VAHTS DNA Clean Beads (N411-02, Vazyme) and elute with 30 μL Milli-Q water according to the manufacturer’s instructions; 6.2. To remove biotin from unligated DNA ends, the reaction mixture (5 μg DNA, 5 μL 10× NEB buffer 2.1, 0.125 μL 10 mM dATP, 0.125 μL 10 mM dGTP, 5 μL T4 DNA polymerase (3000 U / ml, M0203L, NEB) was incubated at 20°C for 4 h; 6.3. Purify DNA again using VAHTS DNA Clean Beads; 6.4. Finally, the VAHTS Universal DNA Library Preparation Kit (ND610-01, Vazyme) was used to prepare sequencing libraries for the Illumina V4 platform, and sequencing was performed on the Illumina platform using the TruSeq technology.

[0033] 7. Protein spectrum analysis Add 1U / mL benzonase nuclease (manufacturer: Millipore, catalog number: 70664) to product II, incubate at 37°C for 30 min, remove nucleic acids in the system, centrifuge at 13000g for 10 min, collect the supernatant, and send the supernatant to BGI Protein Spectrometry for protein spectrum detection.

[0034] The flow chart of the present invention is shown in Figure 1 .

[0035] Comparative Example 1 The preliminary steps refer to the process of steps 1 and 2 in Example 1.

[0036] The difference from Example 1 is: 3.1. The approximately 1×10 7 The cells were resuspended in 1 mL of 4°C 1× lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630), and 10 μL of protease inhibitor (ThermoFisher, 78444) was added. After incubation on ice for 15 min, the cells were centrifuged at 10,000 g for 5 min, and the supernatant was discarded. 3.2. Resuspend the cells again with 100 μL 1× lysis buffer, add 0, 1, 5, 10, and 20 U of micrococcal nuclease to the cells, digest at 37°C for 1 min, and then immediately place on ice; 3.3. Take out 10 μL of product from each of the above 5 samples, add 5 μL of proteinase K and 35 μL of 1× PBS buffer, incubate at 65°C for 30 min, and perform gel electrophoresis.

[0037] The remaining 90 μL sample in step 3.3 was treated at 65°C for 10 min, centrifuged at 12000 g for 10 min, and the supernatant was discarded; 3.4. Resuspend the cells with 100 μL end repair system (10 μL 10×NEB buffer 2.1, 2 μL 100 mM ATP, 5 μL 100 mM DTT, 68 μL water, 5 μL 10 U / μL T4 PNK) and incubate at 37°C for 15 min. 3.5. Then add 10 µL 5U / µL DNA polymerase large fragment Klenow to the product of step 3.4 and incubate at 37°C for 15 min; 3.6. Add the end labeling system (10µL 1mM Biotin-dATP, 10µL 1mM Biotin-dCTP, 1µL 10mM dTTP+dGTP, 5µL 10×T4 DNA ligase buffer, 24µL water) to the system in step 3.5, incubate at 25℃ for 45min, then incubate at 65℃ for 30min; 3.7. Centrifuge the product at 12000g for 10 min, discard the supernatant, and wash once with 1× PBS; 3.8. Add proximity ligation system (425 µL water, 50 µL 10×T4 DNA ligase buffer, 25 µL 400 U / µL T4 DNA ligase) for ligation reaction at 25°C for 3 h; 3.9. Take out 10µL of the product respectively, add 5µL of proteinase K and 35µL of 1× PBS buffer, incubate at 65℃ for 30min, and perform gel electrophoresis.

[0038] Comparative Example 2 The preliminary steps refer to the process of steps 1 and 2 in Example 1.

[0039] The difference from Example 1 is: 3.1. The approximately 1×10 7 The cells were resuspended in 1 mL of 4°C 1× lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630), and 10 μL of protease inhibitor (ThermoFisher, 78444) was added. After incubation on ice for 15 min, the cells were centrifuged at 10,000 g for 5 min, and the supernatant was discarded. 3.2. Resuspend the cells again with 100 μL 1× lysis buffer, add 0, 1, 5, 10, and 20 U of micrococcal nuclease to the cells, digest at 37°C for 1 min, and then immediately place on ice; 3.3. Take out 10 μL of product from each of the above 5 samples, add 5 μL of proteinase K and 35 μL of 1× PBS buffer, incubate at 65°C for 30 min, and perform gel electrophoresis.

[0040] The remaining 90 μL sample in step 3.3 was treated at 65°C for 10 min, centrifuged at 12000 g for 10 min, and the supernatant was discarded; 3.4. Resuspend the cells with 100 μL end repair system (10 μL 10×NEB buffer 2.1, 2 μL 100 mM ATP, 5 μL 100 mM DTT, 68 μL water, 5 μL 10 U / μL T4 PNK) and incubate at 37°C for 15 min. 3.5. Then add 10 µL 5U / µL DNA polymerase large fragment Klenow to the product of step 3.4 and incubate at 37°C for 15 min; 3.6. Add the end labeling system (10µL 1mM Biotin-dATP, 10µL 1mM Biotin-dCTP, 1µL 10mM dTTP+dGTP, 5µL 10×T4 DNA ligase buffer, 24µL water) to the system in step 3.5, incubate at 25℃ for 45min, then incubate at 65℃ for 30min; 3.7. Add 50 µL 1% SDS to the product and resuspend it. Treat it at 65°C for 10 min, then centrifuge it at 12,000 g for 10 min, and wash it once with 1× PBS. 3.8. Add proximity ligation system (425 µL water, 50 µL 10×T4 DNA ligase buffer, 25 µL 400 U / µL T4 DNA ligase) for ligation reaction at 25°C for 3 h; 3.9. Take out 10µL of the product respectively, add 5µL of proteinase K and 35µL of 1× PBS buffer, incubate at 65℃ for 30min, and perform gel electrophoresis.

[0041] Result analysis: according to Figure 2 The results show that the DNA bands without enzyme digestion are single and have high integrity, indicating that the DNA is not degraded; the DNA digested by DpnII shows diffuse bands, indicating that the digestion is sufficient; the size of the product after proximity ligation is higher than that after enzyme digestion, indicating that the ligation is sufficient and efficient.

[0042] according to Figure 3 The results showed that the cell nuclei were broken using different ultrasonic times, and the DNA bands showed different sizes. Among them, the library constructed by ultrasonication for 12 minutes had a higher concentration and could be used for subsequent sequencing analysis.

[0043] according to Figure 4 The results show that Figure 4 A in the figure indicates that the interaction within chromatin reaches 90%, which is much higher than the interaction between chromosomes. The proportion of long-distance interaction (>10kb) within chromatin is about 50%, indicating that the library meets the quality control standards of chromatin high-level structure. Figure 4The results in B show that the DisP-HiC method was used to perform three repetitive experiments, and the data were highly correlated, indicating that the method has good reproducibility and stable technology; Figure 4 Figure C provides a visual analysis of the data, which has low background and high resolution, indicating that the technology has been successfully established and can be used for subsequent analysis.

[0044] according to Figure 5 The results show that the proportion of disordered domains in the K562 proteome and proteins containing disordered domains of >100 amino acids were significantly higher than the random data of the human proteome, and DisP-seq was used as a reference for the published proteome data (literature source DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins). This shows that the method of the present invention can effectively enrich K562 disordered proteins using biotinylated isoxazole.

[0045] according to Figure 6 The results show that Figure 6 A in the figure shows that as the dosage of micrococcal nuclease increases, the DNA fragments gradually become smaller; Figure 6 B in the figure indicates that the DNA after enzyme digestion is subjected to proximity ligation, but the length of the DNA does not increase significantly, indicating that the efficiency of proximity ligation in this process is low.

[0046] according to Figure 7 As a result, the data obtained from the process of comparative example 1 were analyzed. Figure 7 Panel A in the figure shows that the proportion of read pairs generated by sequencing that are aligned to the human hg19 reference genome is high, and the proportion of repeated read pairs within the library is low; Figure 7 Figure B shows the number of read pairs aligned to the reference genome. The intra-chromosomal interaction ratio is higher than the inter-chromosomal interaction ratio, but the number of long-distance interaction read pairs is small, indicating that the experimental neighbor connection efficiency is not sufficient. Figure 7 Figure C visualizes the data, but has high background noise and low resolution, making it unsuitable for subsequent analysis.

[0047] according to Figure 8 As a result, the data obtained from the process of Comparative Example 2 were analyzed. Figure 8 Figure A shows that micrococcal nuclease efficiently digests DNA, and the DNA fragments become significantly smaller; Figure 8 Panel B in the figure shows that the DNA product remains unchanged after proximity ligation, indicating that the efficiency of proximity ligation is low.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously enriching disordered proteins and binding to three-dimensional genome structures, characterized in that: The method comprises the following steps: cross-linking and lysing the cells to be tested, extracting and obtaining cell nuclei; using restriction endonuclease Dpn II to perform enzyme digestion, end repair and proximity ligation on the obtained cell nuclei; and ultrasonically disrupting the cell nuclei after proximity ligation to obtain ultrasonic disruption products; The ultrasonic disruption product is divided into two to obtain product I and product II, the product I is enriched with biotinylated isoxazole for the complex of DNA and protein after ultrasonic disruption, the complex of DNA and protein is washed, eluted and treated with protease; after the protease treatment, DNA is extracted and purified, and the purified DNA is subjected to library construction and sequencing; The product II is treated with nuclease, protein is extracted, and then protein spectrum analysis is performed.

2. The method according to claim 1, characterized in that The cells to be tested were cross-linked using formaldehyde and disuccinimidyl glutarate.

3. The method according to claim 1 or 2, characterized in that: The cross-linking comprises the following steps: resuspending the cells to be tested in a 1×PBS solution, adding formaldehyde and incubating for 8 to 15 minutes, and then adding glycine to terminate the reaction; washing with a 1×PBS solution after terminating the reaction, resuspending with a 1×PBS solution containing 3 mM disuccinimidyl glutaric acid, incubating at room temperature for 30 to 50 minutes, and then adding glycine to continue incubation; washing with a 1×PBS solution containing 0.05% bovine serum albumin, adjusting the cell concentration, centrifuging, removing the supernatant, and collecting the cells for cryopreservation in liquid nitrogen.

4. The method according to claim 1, characterized in that The lysis comprises the following steps: resuspending the cross-linked cells in 1× lysis buffer, adding protease inhibitors, incubating on ice for 10 to 20 minutes, centrifuging, and discarding the supernatant to obtain precipitate 1; Wash precipitate 1 with 1×NEB buffer 3.1, centrifuge and discard the supernatant to obtain precipitate 2; resuspend precipitate 2 with 1×NEB buffer 3.1, add 1% SDS and incubate for 8-12 minutes, and terminate the reaction with 10% Triton X-100.

5. The method according to claim 1, characterized in that The end repair reaction solution includes 5-10 μL 10×NEB buffer 3.1, 1-2 μL 10 mM dCTP, 1-2 μL 10 mM dGTP, 1-2 μL 10 mM dTTP, 10-20 μL 1 mM biotin-14-dATP and 8-15 μL 5 U / μL DNA polymerase I large fragment Klenow.

6. The method according to claim 1, characterized in that The proximity ligation reaction solution includes 100-150 μL 10×T4 ligation buffer, 100-150 μL 10% Triton X-100, 40-60 μL T4 DNA ligase and 300-400 μL Milli-Q water.

7. The method according to claim 1, characterized in that The ultrasonically disrupted reaction solution includes an ultrasonic buffer containing 8-15 μL of protease inhibitors; the ultrasonic buffer includes 40-60 mM HEPES, 120-160 mM NaCl, 0.5-2 mM EDTA, 0.5-2% Triton X-100 and 0.05-0.2% SDS.

8. The method according to claim 1, characterized in that: The method for enriching biotinylated isoxazole comprises the following steps: adding 80-120 μM biotinylated isoxazole after ultrasonic treatment, and incubating with rotation at 4° C. for 0.5-2 h.

9. The method according to claim 1, characterized in that: The washing buffer includes 1× protease inhibitor, 0.05-0.2 mM PMSF, 10-30 mM β-mercaptoethanol, 10-30 mM Tris-HCl, 120-180 mM NaCl, 3-8 mM MgCl2, 0.1-0.8% NP-40 and 5-15% glycerol.

10. The method according to claim 1, characterized in that The elution buffer includes 5-15 mM Tris-HCl, 0.05-0.2% SDS, 120-180 mM NaCl and 3-8 mM DTT.

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