A method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures

Abstract

The present invention provides a method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures, which relates to the technical field of molecular biology. The method of the present invention comprises the following steps: after crosslinking cells with formaldehyde and disuccinimidyl glutarate (DSG), performing digestion treatment, end repair and proximity ligation with the restriction endonuclease Dpn II; after ligation, ultrasonically fragmenting the cell nucleus to obtain an ultrasonically fragmented product, dividing the ultrasonically fragmented product into two parts, biotinylating isoxazole to capture the DNA and protein complex with one part of the product, digesting the protein with protease, extracting and purifying the DNA, and constructing a library and sequencing the purified DNA; treating the other part of the product with nuclease, extracting the protein, and then performing proteomic analysis. The present invention can achieve non-destructive detection of intrinsically disordered proteins and their bound chromatin three-dimensional structures throughout the genome, providing an effective way for their structural analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to a method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures. Background Art

[0002] Intrinsically disordered proteins (IDPs) account for one-third of the human proteome. Characterized by the lack of a stable three-dimensional structure under physiological conditions, they play important roles 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 structures, thereby 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 A-A compartment interactions, maintaining the self-renewal of embryonic stem cells (ESCs) while inhibiting their differentiation into neural progenitor cells. In addition, UTX is an important tumor suppressor with strong phase separation ability. When its intrinsically disordered regions (IDRs) are deleted, UTX loses its condensation ability, leading to tumorigenesis.

[0003] Currently, there are mainly two strategies to study the relationship between IDPs and three-dimensional chromatin structures. The Casl-Drop system allows the induction of liquid condensates by controlling IDPs at specific genomic loci, enabling the observation of changes in three-dimensional chromatin structures. Another method can disrupt the characteristics of intracellular phase separation by combining with 1,6-hexanediol, and detect the impact of phase separation on chromatin three-dimensional structure changes through different time treatments. However, this method cannot understand the changes in the relationship between intrinsically disordered proteins and three-dimensional chromatin structures in the original state within cells. Therefore, there is still a lack of a method that can achieve non-destructive, genome-wide detection of intrinsically disordered proteins and three-dimensional chromatin structures.

[0004] Biotinylated isoxazole (b-isox) is a small molecule that can enrich a broad spectrum of intrinsically 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-separating proteins in Arabidopsis thaliana and rice. In addition, in 2023, the DisP-seq method using the combination of b-isox precipitation and next-generation sequencing provided new insights into protein-DNA interactions. This method identified the one-dimensional genomic characteristics of the binding of intrinsically disordered proteins, but this technology failed to identify the three-dimensional chromatin structure characteristics of intrinsically disordered proteins. Summary of the Invention

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

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures, comprising the following steps: crosslinking and lysing the cells to be tested, and extracting the cell nuclei; subjecting the obtained cell nuclei to enzymatic digestion, end repair and proximity ligation using the restriction endonuclease Dpn II; ultrasonically fragmenting the cell nuclei after the proximity ligation treatment to obtain an ultrasonically fragmented product; dividing the ultrasonically fragmented product into two parts to obtain product I and product II, enriching the complex of DNA and protein after ultrasonic fragmentation with biotinylated isoxazole for the product I, washing, eluting and subjecting the complex of DNA and protein to protease treatment; extracting and purifying DNA after protease treatment, and constructing a library and sequencing the purified DNA; subjecting the product II to nuclease treatment, extracting proteins, and then performing proteomic analysis.

[0008] Preferably, formaldehyde and disuccinimidyl glutarate are used to crosslink the cells to be tested.

[0009] More preferably, the crosslinking comprises the following steps: resuspending the cells to be tested in 1×PBS solution, adding formaldehyde and incubating for 8-15 min, then adding glycine to terminate the reaction; after terminating the reaction, washing with 1×PBS solution, resuspending with 1×PBS solution containing 3 mM disuccinimidyl glutarate, incubating at room temperature for 30-50 min, and then adding glycine and continuing to incubate; washing with 1×PBS solution containing 0.05% bovine serum albumin, adjusting the cell concentration, centrifuging, removing the supernatant, and collecting the cells and freezing them in liquid nitrogen for storage.

[0010] Preferably, the lysis comprises the following steps: resuspending the crosslinked cells in 1× lysis buffer, adding a protease inhibitor, incubating on ice for 10-20 min, then 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.

[0011] Preferably, the reaction solution for end repair includes 5 - 10 μL of 10× NEB Buffer 3.1, 1 - 2 μL of 10 mM dCTP, 1 - 2 μL of 10 mM dGTP, 1 - 2 μL of 10 mM dTTP, 10 - 20 μL of 1 mM biotin-14-dATP, and 8 - 15 μL of 5 U / μL large fragment of DNA polymerase I (Klenow).

[0012] Preferably, the reaction solution for proximity ligation includes 100 - 150 μL of 10× T4 ligation buffer, 100 - 150 μL of 10% Triton X-100, 40 - 60 μL of T4 DNA ligase, and 300 - 400 μL of Milli-Q water.

[0013] Preferably, the reaction solution for sonication includes sonication buffer containing 8 - 15 μL of protease inhibitor; the sonication 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.

[0014] Preferably, the method for biotinylated isoxazole enrichment includes the following steps: adding 80 - 120 μM of biotinylated isoxazole after sonication treatment, and rotating and incubating at 4°C for 0.5 - 2 h.

[0015] Preferably, 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.

[0016] Preferably, the elution buffer includes 5 - 15 mM Tris-HCl, 0.05 - 0.2% SDS, 120 - 180 mM NaCl, and 3 - 8 mM DTT.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discloses a method for simultaneously enriching disordered proteins and their bound three-dimensional genomic structures (DisP-HiC, Disordered protein——High-throughput chromosome conformation capture). In the present invention, formaldehyde and disuccinimidyl glutarate (DSG) are used to crosslink cells to fix the three-dimensional structural characteristics of chromatin; the ligation system and digestion system are further optimized to improve the ligation efficiency; the ligated product is sonicated and the sonicated product is collected. The product is divided into two parts. One part of the product uses biotinylated isoxazole to capture the DNA-protein complex, the protein is digested with protease, and finally the DNA is library constructed and sequenced; the other part of the product is treated with nuclease, the protein is extracted, and proteomic analysis is performed. The present invention can simultaneously enrich disordered proteins and the three-dimensional structures mediated and / or bound by them, and achieve non-destructive detection of the three-dimensional chromatin structures bound by disordered proteins throughout the genome, providing an effective approach for studying the three-dimensional chromatin structural characteristics mediated by disordered proteins throughout the genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the experimental process of the DisP-HiC method of the present invention;

[0020] Figure 2 It is the gel electrophoresis detection of the product after Dpn II digestion and the product after proximity ligation of the DisP-HiC method of the present invention;

[0021] Figure 3 It is the sonication time for obtaining the highest DNA concentration after sonicating the cell nucleus with different sonication times in the DisP-HiC method of the present invention;

[0022] Figure 4 It is the data analysis result of the DisP-HiC method of the present invention. In the figure, A is the statistical ratio of the read pairs of the intra-chromosomal / inter-chromosomal interaction sequences in the DisP-HiC data and the ratio of the interactions with a distance greater than / less than 10,000 base pairs, B is the calculation of the correlation of the results of three repeated experiments, and C is the heat map drawn according to the intra-chromosomal / inter-chromosomal interactions. 1, 2, 3...X respectively represent chromosome numbers;

[0023] Figure 5 It is the enrichment of the K562 cell proteome using biotinylated isoxazole in the DisP-HiC method of the present invention;

[0024] Figure 6 It is a diagram showing the experimental results based on micrococcal nuclease digestion. In the figure, A is the electrophoresis diagram of digesting the cell nucleus with different enzyme dosages for 1 minute, and B is the result of proximity ligation after digestion;

[0025] Figure 7 It is a quality control chart of the data after library construction and sequencing based on micrococcal nuclease digestion. A is the statistical alignment / misalignment to the genomic sequence and the paired reads of repetitive / non-repetitive alignment sequences. B is the proportion of inter-chromosomal / intra-chromosomal sequences and different-sized inserted fragments. C is a heat map drawn based on the interaction between different intra-chromosomes / chromatin after micrococcal nuclease digestion. In the figure, 1, 2, 3... X represent chromosome numbers respectively;

[0026] Figure 8 It is an optimized proximity ligation process based on micrococcal nuclease digestion experiment. In the figure, A is the electrophoresis map of the nuclear digestion for 1 minute using different enzyme dosages. B is the electrophoresis map of the proximity ligation product after the product of digestion is treated with sodium dodecyl sulfate. Specific implementation mode

[0027] The present invention provides a method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures, including the following steps: cross-linking and lysing the cells to be tested, and extracting the nuclei; digesting, end-repairing and performing proximity ligation on the obtained nuclei using the restriction endonuclease Dpn II; ultrasonically fragmenting the nuclei after proximity ligation treatment to obtain an ultrasonically fragmented product; dividing the ultrasonically fragmented product into two parts to obtain product I and product II. Product I is used to enrich the complex of DNA and protein after ultrasonic fragmentation with biotinylated isoxazole, and the complex of DNA and protein is washed, eluted and protease-treated; after protease treatment, DNA is extracted and purified, and the purified DNA is subjected to library construction and sequencing; product II is treated with nuclease, protein is extracted, and then proteomic analysis is performed.

[0028] In the present invention, formaldehyde and disuccinimidyl glutarate 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 - 15 min, then add glycine to terminate the reaction; after terminating the reaction, wash with 1×PBS solution, resuspend with 1×PBS solution containing 3 mM disuccinimidyl glutarate, incubate at room temperature for 30 - 50 min, and then add glycine and continue to incubate; wash with 1×PBS solution containing 0.05% bovine serum albumin, adjust the cell concentration, centrifuge, discard the supernatant, and collect the cells and freeze them in liquid nitrogen for storage. 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 - 15 min, more preferably incubated at room temperature for 10 min. 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: after adding glycine, incubate at room temperature for 3 - 8 min and additionally incubate on ice for 10 - 20 min.

[0029] In the present invention, after cell crosslinking, the cells are lysed. The lysis preferably includes the following steps: Resuspend the crosslinked cells in 1× lysis buffer and add a protease inhibitor, incubate on ice for 10 - 20 min and then centrifuge to discard the supernatant to obtain pellet 1; Wash pellet 1 with 1× NEB buffer 3.1, centrifuge and discard the supernatant to obtain pellet 2; Resuspend pellet 2 with 1× NEB buffer 3.1, add 1% SDS and incubate for 8 - 12 min, and terminate the reaction with 10% Triton X-100. In the present invention, the lysis buffer preferably includes 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630. In the present invention, treatment with sodium dodecyl sulfate (SDS) can preferably make the enzymatic digestion more complete and improve the ligation efficiency.

[0030] In the present invention, after cell lysis, the cell nuclei are extracted, and the cell nuclei are subjected to enzymatic digestion, end repair, and proximity ligation. The specific steps are preferably as follows: After cell lysis, preferably 10-15 μL of 10× NEB buffer 3.1 and 400 U of restriction endonuclease Dpn II are added, and after gentle mixing, digestion is carried out overnight on a shaker at 37 °C; incubation is carried out at 60-68 °C for 15-25 min to inactivate Dpn II, and then cooling is carried out on ice for end repair and proximity ligation. In the present invention, the reaction solution for end repair preferably includes 5-10 μL of 10× NEB buffer 3.1, 1-2 μL of 10 mM dCTP, 1-2 μL of 10 mM dGTP, 1-2 μL of 10 mM dTTP, 10-20 μL of 1 mM biotin-14-dATP, and 8-15 μL of 5 U / μL large fragment Klenow of DNA polymerase I; more preferably, it includes 6 μL of 10× NEB buffer 3.1, 1.5 μL of 10 mM dCTP, 1.5 μL of 10 mM dGTP, 1.5 μL of 10 mM dTTP, 15 μL of 1 mM biotin-14-dATP, and 10 μL of 5 U / μL large fragment Klenow of DNA polymerase I. The reaction solution for proximity ligation preferably includes 100-150 μL of 10× T4 ligation buffer, 100-150 μL of 10% Triton X-100, 40-60 μL of T4 DNA ligase, and 300-400 μL of Milli-Q water; more preferably, it includes 120 μL of 10× ligation buffer, 120 μL of 10% Triton X-100, 50 μL of T4 DNA ligase, and 375 μL of Milli-Q water.

[0031] In the present invention, after enzymatic digestion, end repair, and proximity ligation of the cell nuclei, sonication is carried out. The specific steps of the sonication are preferably as follows: Washing is carried out twice with sonication buffer containing 8-15 μL of protease inhibitor (Thermo Fisher, 78438), and the supernatant is discarded to obtain a precipitate; the precipitate is resuspended in sonication buffer and transferred to a microTUBE-130, and sonication is carried out using a Covaris M220 sonicator to shear the chromatin. In the present invention, the parameter settings of the Covaris M220 sonicator are preferably 75 W, 5% duty cycle, 200 pulses / burst, and 12 min. In the present invention, after sonication treatment, centrifugation is preferably carried out at 12000 g for 8-15 min, and the supernatant is collected; the supernatant is made up to volume with sonication buffer.

[0032] In the present invention, an ultrasonic fragmentation product is obtained after ultrasonic fragmentation treatment; the ultrasonic fragmentation product is divided into two parts to obtain Product I and Product II. Product I is used to enrich the complex of DNA and protein after ultrasonic fragmentation. 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 subjected to library construction and sequencing. The specific steps are preferably as follows: Biotinylated isoxazole (b-isox) is added to the above-mentioned fixed-volume supernatant to obtain a mixture, and the mixture is rotated and incubated at 4 °C for 0.5 - 2 h, and then centrifuged at 13000 g for 10 - 20 min to remove the supernatant and collect the precipitate; the precipitate is washed twice with the washing buffer, and then centrifuged at 4 °C and 13000 g for 10 - 20 min to remove the supernatant and collect the precipitate; the precipitate is resuspended in the elution buffer, and RNase A is added and shaken and incubated at 37 °C at 600 rpm for 25 - 35 min. Then, proteinase K is added and shaken overnight at 60 - 70 °C at 600 rpm. 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, and 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 all be obtained by purchasing through conventional channels.

[0033] In the present invention, after enriching intrinsically disordered proteins using biotinylated isoxazole, DNA is extracted and purified, and the purified DNA is used to construct a library for 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; to remove the biotin at the 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) is incubated at 18-25 °C for 3-4 min; the DNA is purified again using VAHTS DNA Clean Beads; finally, a sequencing library is prepared for the Illumina V4 platform using the VAHTS Universal DNA Library Preparation Kit (ND610-01, Vazyme), and sequencing is performed on the Illumina platform using TruSeq technology.

[0034] In the present invention, Product II is treated with nuclease to extract proteins, and then subjected to proteomic analysis. The specific steps are preferably as follows: Benzonase nuclease is added to Product II and incubated at 35-37 °C for 25-35 min. After removing the nucleic acids in the system, centrifugation is performed at 13,000 g for 8-15 min, and the supernatant is collected. The supernatant is subjected to proteomic detection. In the present invention, the Benzonase nuclease is obtained by purchasing through conventional channels. The Benzonase nuclease described in the present invention is purchased from Merck Millipore, and the product number is 70664. The present invention preferably sends the supernatant to be subjected to proteomic detection to BGI Proteomics for detection and analysis.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0037] Example 1

[0038] A method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures, the specific steps are as follows:

[0039] 1. Cell culture

[0040] 1.1. The K562 human chronic myeloid leukemia cell line (ATCC, CRL-3344) was cultured using 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), and incubated in a 5% CO2, 37 °C constant temperature incubator. For subculture, 3.5×10 5 cells were seeded into 10 mL of complete medium in a 10 cm culture dish every 2 - 3 days.

[0041] 2. Cell crosslinking

[0042] 2.1. The complete medium containing K562 human chronic myeloid leukemia cells was centrifuged at 800 g for 5 min, and the cultured K562 cells were collected and washed once by resuspending them completely with 1× PBS at room temperature;

[0043] 2.2. The cells were resuspended in 1× PBS at a concentration of 1×10 6 cells / mL;

[0044] 2.3. To fix the cells, 37% formaldehyde was added to a final concentration of 1%, and the mixture was gently incubated at room temperature for 10 min;

[0045] 2.4. 2.5 M glycine was added to a final concentration of 168 mM to terminate the formaldehyde reaction, and then the mixture was incubated at room temperature for 5 min and additionally incubated on ice for 15 min;

[0046] 2.5. Centrifuge at 800 g for 5 min and wash twice with 1× PBS at room temperature;

[0047] 2.6. Resuspend in 1× PBS at room temperature containing 3 mM disuccinimidyl glutarate (DSG) and incubate with mixing at room temperature for 45 min;

[0048] 2.7. Add 2.5 M glycine to a final concentration of 0.4 M, then incubate at room temperature for 5 min and additionally incubate on ice for 15 min;

[0049] 2.8. Wash twice with 4 °C 1× PBS containing 0.05% bovine serum albumin (BSA);

[0050] 2.9. Adjust the concentration to 1×10 7cells / mL, and then aliquot the cells into 1.5 mL low-attachment Eppendorf EP tubes, centrifuge at 2500 g for 5 min, and remove the supernatant;

[0051] 2.10. The cells are quickly frozen in liquid nitrogen and stored at -80 °C.

[0052] 3. Cell lysis and chromatin digestion

[0053] 3.1. Resuspend approximately 1×10 7 cells obtained in step 2 in 1 mL of 4 °C 1× lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630), add 10 μL of protease inhibitor (ThermoFisher, 78444), incubate on ice for 15 min, then centrifuge at 2500 g for 5 min, and discard the supernatant;

[0054] 3.2. Wash twice with 500 μL of 4 °C 1× NEB buffer 3.1, centrifuge at 2500 g for 5 min, and discard the supernatant;

[0055] 3.3. Resuspend the pellet in 342 μL of 1× NEB buffer 3.1, add 38 μL of 1% SDS, incubate at 65 °C for 10 min, and then terminate the reaction with 43 μL of 10% Triton X-100;

[0056] 3.4. Add 12 μL of 10× NEB buffer 3.1 and 400 U of Dpn II, mix gently, and digest overnight on a shaker at 37 °C.

[0057] 4. End repair and proximity ligation;

[0058] 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;

[0059] 4.2. Prepare the reaction mixture for end repair, which includes 6 μL of 10× NEB buffer 3.1, 1.5 μL of 10 mM dCTP, 1.5 μL of 10 mM dGTP, 1.5 μL of 10 mM dTTP, 15 μL of 1 mM biotin-14-dATP, and 10 μL of 5 U / μL large fragment of DNA polymerase I Klenow;

[0060] 4.3. Add 60 μL of the end-repair reaction solution to the digested cell nuclei and incubate at 23 °C for 4 h. After cooling, add the proximity ligation reaction solution (120 μL of 10× T4 ligation buffer, 120 μL of 10% Triton X-100, 50 μL of T4 DNA ligase, 375 μL of Milli-Q water), mix, and incubate at 16 °C for 4 h;

[0061] 5. Nucleus sonication and biotinylated isoxazole enrichment

[0062] 5.1. Centrifuge the product after proximity ligation in step 4 at 2800 g for 10 min and discard the supernatant. Wash twice with 1 mL of sonication 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;

[0063] 5.2. Resuspend the precipitate in 130 μL of sonication buffer and transfer it to a microTUBE-130;

[0064] 5.3. Perform chromatin shearing using a Covaris M220 sonicator (75 W, 5% duty cycle, 200 pulses / burst, 12 min);

[0065] 5.4. Centrifuge at 12000 g for 10 min and collect the supernatant; adjust the supernatant to 600 μL using sonication buffer;

[0066] 5.5. Divide the 600 μL of supernatant obtained in step 5.4 into two equal parts to obtain product I and product II; add 100 μM biotinylated isoxazole to product I, and incubate the mixture with rotation at 4 °C for 1 h;

[0067] 5.6. Centrifuge at 13000 g at 4 °C for 15 min, remove the supernatant and collect the precipitate, wash the precipitate twice with washing buffer, centrifuge at 13000 g at 4 °C for 15 min, remove the supernatant and collect the precipitate; the washing buffer composition contains 20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM MgCl2; 0.5% NP-40; 10% glycerol, plus 1× protease inhibitor, 0.1 mM PMSF, and 20 mM β-mercaptoethanol;

[0068] 5.7 Resuspend the precipitate collected in step 5.6 in 200 μL of elution buffer (10 mM Tris-HCl, pH 8.0; 0.1% SDS; 150 mM NaCl; 5 mM DTT), add 1 μL of RNase A and incubate with shaking at 600 rpm at 37 °C for 30 min. Then, add 10 μL of proteinase K and shake overnight at 65 °C at 600 rpm.

[0069] 6. DNA Purification and Library Preparation

[0070] 6.1 Extract DNA using 2×VAHTS DNA Clean Beads (N411-02, Vazyme) and elute with 30 μL of Milli-Q water according to the manufacturer's instructions;

[0071] 6.2 To remove the biotin at the unligated DNA ends, incubate the reaction mixture (5 μg DNA, 5 μL of 10×NEB buffer 2.1, 0.125 μL of 10 mM dATP, 0.125 μL of 10 mM dGTP, 5 μL of T4 DNA polymerase (3000 U / ml, M0203L, NEB)) at 20 °C for 4 h;

[0072] 6.3 Purify the DNA again using VAHTS DNA Clean Beads;

[0073] 6.4 Finally, prepare a sequencing library for the Illumina V4 platform using the VAHTS Universal DNA Library Preparation Kit (ND610-01, Vazyme) and perform sequencing on the Illumina platform using TruSeq technology.

[0074] 7. Protein Mass Spectrometry

[0075] Add 1 U / mL of benzonase nuclease (manufacturer: Millipore, catalog number: 70664) to Product II, incubate at 37 °C for 30 min, remove the nucleic acids in the system, and then centrifuge at 13000 g for 10 min to collect the supernatant. Send the supernatant to BGI Protein Mass Spectrometry Company for protein mass spectrometry detection.

[0076] For the flow chart of the present invention, see Figure 1 .

[0077] Comparative Example 1

[0078] Refer to the procedures of steps 1 and 2 in Example 1 for the previous steps.

[0079] The difference from Example 1 is:

[0080] 3.1. Resuspend approximately 1×10 7 cells obtained in step 2 in 1 mL of 1× lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630) at 4°C, and add 10 μL of protease inhibitor (ThermoFisher, 78444). After incubating on ice for 15 min, centrifuge at 10,000 g for 5 min and discard the supernatant;

[0081] 3.2. Resuspend the cells again using 100 μL of 1× lysis buffer. Add 0, 1, 5, 10, 20 U of micrococcal nuclease to the cells respectively, digest at 37°C for 1 min, and then immediately place on ice;

[0082] 3.3. Take 10 μL of the 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.

[0083] For the remaining 90 μL of the sample in step 3.3, treat at 65°C for 10 min, centrifuge at 12,000 g for 10 min, and discard the supernatant;

[0084] 3.4. Resuspend the cells using 100 μL of end repair system (10 μL of 10× NEB buffer 2.1, 2 μL of 100 mM ATP, 5 μL of 100 mM DTT, 68 μL of water, 5 μL of 10 U / µL T4 PNK), and incubate at 37°C for 15 min;

[0085] 3.5. Then add 10 µL of 5 U / µL Klenow fragment of DNA polymerase to the product in step 3.4 and incubate at 37°C for 15 min;

[0086] 3.6. Add the end labeling system (10 µL of 1 mM Biotin-dATP, 10 µL of 1 mM Biotin-dCTP, add 1 µL of 10 mM dTTP + dGTP respectively, 5 µL of 10× T4 DNA ligase buffer, 24 µL of water) to the system in step 3.5, incubate at 25°C for 45 min, and then incubate at 65°C for 30 min;

[0087] 3.7. Centrifuge the product at 12,000 g for 10 min, discard the supernatant, and wash once with 1× PBS;

[0088] 3.8. Add the proximity ligation system (425 µL of water, 50 µL of 10× T4 DNA ligase buffer, 25 µL of 400 U / µL T4 DNA ligase) for ligation reaction at 25°C for 3 h;

[0089] 3.9. Take out 10 µL of the product respectively, add 5 µL of proteinase K, 35 µL of 1×PBS buffer, incubate at 65 °C for 30 min, and perform gel electrophoresis.

[0090] Comparative Example 2

[0091] The procedures in the previous steps refer to the procedures of Step 1 and Step 2 in Example 1.

[0092] The difference from Example 1 is:

[0093] 3.1. Resuspend approximately 1×10 7 cells obtained in Step 2 in 1 mL of 1× lysis buffer at 4 °C (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA-630), add 10 μL of protease inhibitor (ThermoFisher, 78444), incubate on ice for 15 min, centrifuge at 10000 g for 5 min, and discard the supernatant;

[0094] 3.2. Resuspend the cells again with 100 μL of 1× lysis buffer, add 0, 1, 5, 10, 20 U of micrococcal nuclease to the cells respectively, digest at 37 °C for 1 min, and then immediately place on ice;

[0095] 3.3. Take out 10 μL of the product from each of the above 5 samples, add 5 μL of proteinase K, 35 μL of 1×PBS buffer, incubate at 65 °C for 30 min, and perform gel electrophoresis.

[0096] For the remaining 90 μL of the sample in Step 3.3, treat at 65 °C for 10 min, centrifuge at 12000 g for 10 min, and discard the supernatant;

[0097] 3.4. Resuspend the cells with 100 μL of end repair system (10 μL of 10× NEB buffer 2.1, 2 μL of 100 mM ATP, 5 μL of 100 mM DTT, 68 μL of water, 5 μL of 10 U / µL T4 PNK), incubate at 37 °C for 15 min;

[0098] 3.5. Then add 10 µL of 5 U / µL Klenow fragment of DNA polymerase to the product of Step 3.4, incubate at 37 °C for 15 min;

[0099] 3.6. Add the end-labeling system to the system in Step 3.5 (10 µL of 1 mM Biotin-dATP, 10 µL of 1 mM Biotin-dCTP, add 1 µL of 10 mM dTTP + dGTP respectively, 5 µL of 10× T4 DNA ligase buffer, 24 µL of water), incubate at 25 °C for 45 min, and then incubate at 65 °C for 30 min;

[0100] 3.7. Resuspend the product with 50 µL of 1% SDS, treat at 65 °C for 10 min, then centrifuge at 12,000 g for 10 min, and wash once with 1× PBS;

[0101] 3.8. Add the proximity ligation system (425 µL of water, 50 µL of 10× T4 DNA ligase buffer, 25 µL of 400 U / µL T4 DNA ligase) for ligation reaction, at 25 °C for 3 h;

[0102] 3.9. Take out 10 µL of the product respectively, add 5 µL of proteinase K, 35 µL of 1× PBS buffer, incubate at 65 °C for 30 min, and perform gel electrophoresis.

[0103] Result analysis:

[0104] According to Figure 2 the results, the un-digested DNA band is single and has high integrity, indicating that the DNA is not degraded; the DNA after DpnII digestion shows a diffuse band, indicating sufficient digestion; the size of the product after proximity ligation is higher than that of the product after digestion, indicating sufficient and efficient ligation.

[0105] According to Figure 3 the results, when the cell nucleus is broken using different sonication times, the DNA bands show different sizes. Among them, the library concentration is higher when sonicated for 12 min, which can be used for subsequent sequencing analysis.

[0106] According to Figure 4 the results, Figure 4 A in Figure 4 indicates that the interaction within chromatin reaches 90%, which is much higher than the interaction between chromatins. The proportion of long-distance interactions within chromatin (>10 kb) is about 50%; it shows that the library meets the quality control standards of chromatin higher-order structure. Figure 4 Result B in

[0107] indicates that three repeated experiments were carried out using the DisP-HiC method, and the correlation between the data is very high, indicating that this method has good repeatability and stable technology; Figure 5The results show that the proportions of calving in the disordered domains of the K562 proteome and proteins with disordered domains containing >100 amino acids, when statistically analyzed separately, are significantly higher than the random data of the human proteome. DisP-seq uses published proteome data as a reference (literature source: DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins). This indicates that the method of the present invention using biotinylated isoxazole can effectively enrich K562 disordered proteins.

[0108] According to Figure 6 The results show that Figure 6 in A of [reference] shows that as the amount of micrococcal nuclease is increased, the DNA digestion fragments gradually become smaller; Figure 6 in B of [reference] shows that proximity ligation is performed on the digested DNA, but the DNA length does not increase significantly, indicating that the proximity ligation efficiency of this process is low.

[0109] According to Figure 7 The results show that the data obtained from the process of Comparative Example 1 are analyzed. Figure 7 In Figure A of [reference], it shows that the proportion of the read pairs generated by sequencing aligned to the human hg19 reference genome is relatively high, and the proportion of the duplicate read pairs within the library is relatively low; Figure 7 In Figure B of [reference], it shows that for the read pairs aligned to the reference genome, the proportion of the interactions within the chromosome is higher than that between chromosomes, but the number of read pairs for long-distance interactions is small, indicating that the proximity ligation efficiency of the experiment is insufficient; Figure 7 In Figure C of [reference], the data is visualized, with high background noise and low resolution, which is not suitable for subsequent analysis.

[0110] According to Figure 8 The results show that the data obtained from the process of Comparative Example 2 are analyzed. Figure 8 In Figure A of [reference], it shows that micrococcal nuclease efficiently digests DNA, and the DNA fragments become significantly smaller; Figure 8 In Figure B of [reference], it shows that the DNA product remains unchanged after proximity ligation, indicating that the proximity ligation efficiency is low.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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; ultrasonically disrupting the cell nuclei after proximity ligation to obtain ultrasonic disruption products; dividing the ultrasonic disruption products into two to obtain product I and product II, enriching the complex of DNA and protein after ultrasonic disruption with biotinylated isoxazole in product I, washing, eluting and treating the complex of DNA and protein with protease; 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; The cells to be tested were cross-linked using formaldehyde and disuccinimidylglutarate; The cross-linking comprises the following steps: resuspending the cells to be tested in a 1×PBS solution, adding formaldehyde and incubating for 8-15 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 3mM disuccinimidyl glutaric acid, incubating at room temperature for 30-50 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; The enzyme digestion step is as follows: after cell lysis, 10-15 μL of 10×NEB buffer 3.1 and 400U of restriction endonuclease Dpn II are added, 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; 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; The proximity ligation reaction solution includes 100-150 μL 10×T4 ligation buffer, 100-150 μL 10% TritonX-100, 40-60 μL T4 DNA ligase and 300-400 μL Milli-Q water.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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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