Spatial three-dimensional genomics sequencing method based on microfluidic chip and use thereof

Abstract

Disclosed in the present invention are a spatial three-dimensional genomics sequencing method based on a microfluidic chip and the use thereof. According to the method of the present invention, the molecular labeling process of a tissue section is segmented, that is, on the basis of the process of "overall labeling — partial lysis — partial labeling — overall lysis", position labeling at the micron level is firstly performed at the spatial position level on the tissue section, then the tissue section is lysed into protein-nucleic acid complexes at the nanometer level by means of ultrasonic disruption, the macromolecular complexes are individually labeled at the nanometer level, and finally the macromolecular complexes are lysed to obtain DNA molecules containing both spatial position information and chromatin conformation information for library construction and sequencing. The method of the present invention allows for efficient joint analysis of resolution at the visualized micron level and the non-visualized base length level, and thus can be applied to the development or preparation of a product for disease detection or a product for observing organ development.

Description

A spatial three-dimensional genomics sequencing method based on microfluidic chip and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for spatial three-dimensional genomics sequencing based on a microfluidic chip and its application. Background Art

[0002] In life activities, the three-dimensional genome is a very important link besides the transcriptome, and plays a key role in the development of organs, the generation of lymphoma, the occurrence of leukemia, and other processes. Therefore, in-depth exploration of the three-dimensional conformation of chromatin has great biological significance for systematically understanding key biological issues such as disease generation and organ development. So far, although some commercial sequencing methods can complete high-throughput sequencing of spatial transcriptomes, these methods are all concentrated on the spatial transcriptome, and there is no mature system for other omics research. Spatial omics technology at the scientific research level is also in its infancy, and has only made some progress in the genome, proteome, epigenome and other directions. At present, there is still a lack of high-throughput sequencing solutions for three-dimensional genomics.

[0003] Currently, the basic process for adding molecular markers in all existing spatial omics solutions is to first label the entire tissue section with regionalized spatial barcode information, and then lyse the tissue as a whole to obtain the protein or nucleic acid molecules with spatial barcode information. However, this process can only distinguish all target molecules at the submicron level at best, and cannot further improve the resolution.

[0004] SUMMARY OF THE INVENTION

[0005] The main technical problem to be solved by the present invention is how to detect the three-dimensional conformation of chromatin at different spatial positions in tissue sections. In order to solve the above technical problems, the present invention adopts a spatial three-dimensional genomic sequencing method based on a microfluidic chip. The method of the present invention segments the molecular labeling process of tissue sections, that is, through the "overall labeling-partial cleavage-partial labeling-overall cleavage" process, the tissue sections are first marked at the micron level at the spatial position level, and then the tissue sections are cleaved into nanometer-level protein-nucleic acid complexes by ultrasonic crushing and the macromolecular complexes are individually labeled at the nanometer level. Finally, the macromolecular complexes are cleaved and DNA molecules containing both spatial position information and chromatin conformation information are obtained for library construction and sequencing. The method of the present invention can effectively combine the resolution from the visualized micron level and the non-visualized base length level for analysis. Technical issues

[0006] The technical problem to be solved by the present invention is how to detect the three-dimensional conformation of chromatin at different spatial positions in tissue sections and / or how to perform high-throughput spatial three-dimensional genomic sequencing.

[0007] In all existing spatial omics solutions, the basic process for adding molecular markers is to first label the entire tissue section with regional spatial barcode information, and then lyse the tissue as a whole to obtain the protein or nucleic acid molecules with spatial barcode information. However, this process can only distinguish all target molecules at the submicron level at best, and cannot further improve the resolution. Technical Solutions

[0008] In order to solve the above technical problems, the present invention first provides a method for detecting the three-dimensional genome conformation of chromatin space, which may include the following steps:

[0009] A1) Crosslinking and Fixation: After assembling the tissue slice to be tested with the reaction pool of a PDMS (polydimethylsiloxane) chip, a crosslinking agent is added to the reaction pool to perform crosslinking, thereby obtaining the tissue slice to be tested with fixed nucleic acid and protein conformations; the chip structure includes the reaction pool;

[0010] A2) cell permeabilization: permeabilizing the tissue slice to be tested in the reaction cell to obtain a permeabilized tissue slice to be tested;

[0011] A3) Nucleic acid linker ligation: performing double enzyme digestion on the nucleic acid in the permeabilized tissue slice to be tested in the reaction pool and adding a nucleic acid linker to obtain a tissue slice with added nucleic acid linker; separating the tissue slice with added nucleic acid linker from the PDMS chip to obtain a separated tissue slice with added nucleic acid linker;

[0012] A4) Spatial Position Overall Labeling: Assembling the tissue slice with nucleic acid adapters added after separation with a microfluidic chip to obtain an assembled microfluidic chip, wherein the microfluidic chip comprises a sample loading port, a central region, and a sample outlet, wherein the central region of the assembled microfluidic chip is bonded to the tissue slice with nucleic acid adapters added after separation; adding a total of i X-labeled mixed solutions (X1-Xi) to the injection channel and aspirating the mixed solution to the central region using a vacuum pump, thereby adding i barcode labels to the nucleic acids of the tissue slice with nucleic acid adapters added in the X-axis direction in the microfluidic chip. , obtaining an X-axis marked tissue slice; then, adding a total of i types of Y-labeled mixed solutions Y1-Yj to the injection hole channel and sucking the mixed solution to the central area through a vacuum pump, so as to add j types of barcode labels to the nucleic acids in the X-axis marked tissue slice in the Y-axis direction in the microfluidic chip, thereby obtaining a spatially overall marked tissue slice; the X-axis direction is perpendicular to the Y-axis direction; the spatially overall marked tissue slice is marked as i×j pixels, and the nucleic acid in each pixel contains a different combination X of barcodes X and Y. i Y j The X mark and the Y mark have different fluorescent groups, and the different fluorescent groups produce fluorescence of different colors under the action of excitation light; i is a natural number greater than 1 and less than or equal to the number of injection holes, and j is a natural number greater than 1 and less than or equal to the number of injection holes;

[0013] A5) ultrasonically disrupting the spatially labeled tissue section: ultrasonically disrupting the spatially labeled tissue section to obtain a cleavage product containing a protein-nucleic acid complex;

[0014] A6) NHS-activated magnetic bead coupling: coupling the protein-nucleic acid complex in the lysate with NHS-activated magnetic beads to obtain a complex coupled with NHS-activated magnetic beads;

[0015] A7) Partial labeling: The nucleic acid in the complex coupled to the NHS-activated magnetic beads is subjected to the SPLiT-seq method through n rounds of "dispersion-mixing" steps, with k different barcode labels added in each round to obtain k added barcodes. n A complex of tandemly combined barcodes, wherein different nucleic acids of the complex may contain k n different series combination barcodes; n may be a natural number greater than 1 and less than or equal to 5. k may be a natural number greater than 1 and less than or equal to 100;

[0016] A8) Reverse cross-linking to overall cleavage: Add the added k nThe complex of the tandem combination barcode is reverse-crosslinked to obtain a cross-linked nucleic acid fragment, and the cross-linked nucleic acid fragment is purified to obtain a nucleic acid fragment to be sequenced; the nucleic acid fragment to be sequenced contains the nucleic acid adapter sequence, the X-ray diffraction pattern of the spatial position of the nucleic acid fragment to be sequenced on the tissue section to be sequenced, and the like. i Y j The barcode sequence and the k n tandem combination barcode sequences;

[0017] A9) Library construction and sequencing: The nucleic acid fragments to be sequenced are amplified using single-stranded random primers to construct a sequencing library, the sequencing library is sequenced to obtain sequencing data, and the sequencing data is analyzed. i Y j Barcode sequence is used to obtain the three-dimensional genomic conformation of the chromatin space of the tissue section to be tested.

[0018] In the above method, the SPLiT-seq method of adding n rounds of different barcode labels through n rounds of "scatter-mix" steps may include the following steps:

[0019] A7-1) In the first round of reaction, all of the complexes coupled to the NHS-activated magnetic beads are dispersed into a reaction system 1 containing k different barcode tags, and the barcode tags from the first round are added to obtain complexes with k different barcode tags added thereto; and then all of the complexes with k different barcode tags added thereto are mixed to obtain an n1 mixture; wherein the nucleic acid fragments in the n1 mixture have k different barcode tags;

[0020] A7-2) In the second round of reaction, all the complexes with k different barcode tags added in the n1 mixture are redispersed into the reaction system 2 containing k different barcode tags, and k different barcode tags are added in the second round to obtain complexes with k different barcode tags added again. The complexes with k different barcode tags added again are then mixed to obtain the n2 mixture; the nucleic acid fragments in the n2 mixture have k different barcode tags. 2 Different barcode labels;

[0021] A7-3) In the nth round of reaction, the n (n-1) All the complexes with (n-1) rounds of k different barcode labels added to the mixture are dispersed into the reaction system n containing k different barcode labels. The k different barcode labels in the nth round are added to obtain the complexes with k different barcode labels added in the nth round. Then, the complexes with k different barcode labels added in the nth round are mixed to obtain n (n) mixture; n (n) The nucleic acid fragments in the mixture carry k n Different barcode labels.

[0022] The n may be a natural number greater than 1 and less than or equal to 5, and the k may be a natural number greater than 1 and less than or equal to 100.

[0023] In the above method, the reaction cell can be a PDMS (polydimethylsiloxane) single-hole chip, and the central hole size can be 0.7 cm×0.7 cm or 0.7 cm×1.2 cm. In the above method, the microfluidic chip can be a PDMS (polydimethylsiloxane) microfluidic chip.

[0024] In the above method, the double enzyme digestion in A3) can be performed using restriction endonucleases Mse I and HinP1I; the nucleic acid linker in A3) can include a linker for the sticky ends digested by Mse I and a linker for the sticky ends digested by HinP1I.

[0025] In the above method, the Y label in A4) may be modified with a biotin group. The purification in A8) may be performed by using magnetic beads with streptavidin to form a complex with the biotin group for adsorption purification to obtain the nucleic acid fragment to be sequenced.

[0026] In the above method, the ultrasonic crushing in A5) is non-contact ultrasonic crushing, and the time of the non-contact ultrasonic crushing can be greater than or equal to 4 minutes and less than or equal to 10 minutes.

[0027] In the above method, the single-ended random primer amplification may include the following steps: using a single-stranded random primer containing six random bases N at the 3' end and a sequencing adapter sequence at the 5' end to randomly bind to the nucleic acid fragment to be sequenced by annealing and then annealing to obtain a annealed product, and then using a DNA polymerase to completely amplify the annealed product to obtain the sequencing library containing two sequencing adapter sequences. The sequence length of the sequencing library can be 350 to 750 bp. The DNA polymerase can be a DNA polymerase Klenow fragment with strand displacement activity.

[0028] The N may be any one of A, T, C or G.

[0029] In order to solve the above technical problems, the present invention also provides a method for spatial three-dimensional genomic sequencing, characterized in that: the method may include using the sequencing library construction method described above to obtain a spatial three-dimensional genomic sequencing library of the tissue section to be tested, and sequencing the sequencing library.

[0030] The method for constructing the sequencing library described above also falls within the scope of protection of the present invention.

[0031] In order to solve the above technical problems, the present invention also provides the application of the method described above in developing or preparing a product for disease detection.

[0032] The application of the above-described method in developing or preparing products for observing organ development also falls within the scope of protection of the present invention.

[0033] The present invention also provides a method for diagnosing a subject, comprising sequencing a tissue section of the subject using the spatial three-dimensional genomics sequencing method described above, and then analyzing the sequencing data to diagnose the tissue section of the subject.

[0034] In this application, "subject" includes a person who is being diagnosed with a disease or preventing a disease as a patient. The methods described herein can be used to diagnose animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorphs (order Logomorpha) mammals, such as rabbits. The mammal can be a carnivora (order Carnivora), including felines (cats) and canines (dogs). The mammal can be an artiodactyla (order Artiodactyla), including bovines (cows) and suids (pigs), or a perssodactyla (order Perssodactyla), including equines (horses). The mammal can be a primate (order Primate), a ceboid (order Ceboid) or a simian (order Simoid) (monkeys) or an anthropoid (order Anthropoid) (humans and apes). In some embodiments, the mammal is a human.

[0035] The method of the present invention innovatively segments the molecular labeling process of tissue sections. First, the tissue sections are spatially labeled at the micrometer level. Then, the tissue sections are ultrasonically fragmented into nanometer-scale protein-nucleic acid complexes, and the macromolecular complexes are individually labeled at the nanometer level. Finally, the macromolecular complexes are fragmented to obtain DNA molecules containing both spatial position information and chromatin conformation information. Through this "global labeling-partial fragmentation-partial labeling-global fragmentation" process, the present invention can effectively combine analysis at the visual micrometer level with the non-visual base length level, achieving a technological breakthrough that is currently unattainable. Beneficial effects

[0036] The present invention has developed a method for sequencing three-dimensional genomes at the spatial level of tissue sections. This technical solution has the following advantages:

[0037] ①. The method of the present invention realizes the detection of spatial three-dimensional genome for the first time. Compared with the three-dimensional genome at the tissue level and single-cell level, it can provide spatial position information within the tissue and provide a new technical platform for chromatin biology.

[0038] ②. Both spatial resolution and coverage area have been improved, covering an area of ​​5mm×5mm to 10mm×5mm. It can be used to detect intact mouse brain tissue slices and embryonic tissue slices within 16 days of development. The area of ​​each detection point is 50μm×50μm to 25μm×25μm.

[0039] ③. The method of the present invention adopts a multi-end interaction detection method (SPLIT tag combination), which can detect a larger number of intermolecular interactions compared to the double-end detection Hi-C experimental scheme.

[0040] ④. Compared with previous tissue-level and single-cell-level methods, the method of the present invention can detect more inter-chromosomal interactions, providing technical support for analyzing the contribution of chromosome territory to tissue and organ development and cell fate determination.

[0041] ⑤. The method of the present invention does not require sophisticated instruments and equipment, the overall process cost is low, and the instrument molds can be reused. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows schematic diagrams of the assembly structures of a tissue slice and a PDMS chip, and a tissue slice and a PDMS microfluidic chip. A shows the assembly structure of a tissue slice and a PDMS reaction pool chip. B shows the assembly structure of a tissue slice and a PDMS microfluidic chip (96-well, 25μm resolution), including the central area, sample loading port, and sample outlet. C shows the assembly structure of a tissue slice and a PDMS microfluidic chip (96-well, 50μm resolution), including the central area, sample loading port, and sample outlet.

[0043] Figure 2 shows fluorescence detection images after labeling the X- and Y-axes with fluorescent barcode primers. The top image shows a 100-well 25μm resolution reaction set-up. The left image shows fluorescence detection images after labeling the X-axis with fluorescent barcode primers, and the right image shows fluorescence detection images after labeling the Y-axis with fluorescent barcodes. The bottom image shows a 50-well 50μm resolution reaction set-up. The left image shows fluorescence detection images after labeling the X-axis with fluorescent barcode primers, and the right image shows fluorescence detection images after labeling the Y-axis with fluorescent barcodes.

[0044] FIG3 is a schematic diagram of the overall process of the method of the present invention.

[0045] Figure 4 shows the quality control results of the library after gel recovery and fragment sorting. The horizontal and vertical axes represent the proportions of DNA fragments of different lengths, respectively.

[0046] Figure 5 shows the library balance results obtained using pair-end sequencing and the evaluation of the number of tags connected to all sequencing reads. The upper figure shows the library balance results. The horizontal and vertical axes are the distribution ratios of the four bases at different positions of the 300bp bases on both ends. The higher the degree of dispersion of the base distribution, the worse the balance of this site. Since the barcode primers added in each round are composed of an identical 7bp linker sequence and a specific 14bp barcode sequence, and the proportion of single bases in the linker sequence is high, the base balance of the site is poor. Therefore, the base balance of the linker region can be used to determine the proportion of sequences that are correctly connected to the linker sequence. The lower figure is an evaluation of the number of tags connected to all reads, and the vertical axis is the proportion of reads.

[0047] Figure 6 shows a heat map of inter-chromosome interactions and a heat map of intra-chromosome interactions for all points in space. The left figure shows a heat map of inter-chromosome interactions for all points in space, with the horizontal and vertical axes representing different chromosome segments, and the heat map indicates the strength of interactions between segments. The right figure shows a heat map of intra-chromosome interactions for all points in space, with the horizontal and vertical axes representing different chromosome segments, and the heat map indicates the strength of interactions between segments.

[0048] Figure 7 shows the number of reads, clusters, and interactions across all sites in space. The left panel shows the number of reads across all sites in space, the middle panel shows the number of clusters across all sites in space, and the right panel shows the number of interactions across all sites in space. The horizontal and vertical axes correspond to the horizontal and vertical coordinates of the sites in space, respectively.

[0049] Figure 8 shows the results of using the k-means algorithm to map to the UMAP two-dimensional space (left) and embryo space (right). Different colors in the figure represent different categories of organs or tissues in the space.

[0050] FIG9 shows the size of protein-DNA complexes after contact sonication using a time gradient of 15 s, 40 s, and 1 min.

[0051] Figure 10 shows the results of a comparative experiment testing DNA digestion methods. The left image shows the result of restriction endonuclease digestion and fluorescent primer ligation, followed by fluorescence microscopy. The fluorescent area represents the cell nucleus. The right image shows the result of DNase I digestion and fluorescent primer ligation, followed by fluorescence microscopy using the same parameters. Clear signals are observed in all tissue regions.

[0052] Figure 11 shows the results of a random sampling of some sequences from this sequencing run. The boxed area is where the empty adapters are connected. The sequences in the figure correspond from top to bottom to sequences 731 to 758 in the sequence listing.

[0053] Figure 12 shows the statistical data obtained from experiments using different ultrasound modes, different ultrasound intensities, and different ultrasound durations. A shows the ratio of left and right chromosomes within chromosomes and between chromosomes (ordinate) obtained using contact ultrasound at 30W and 40W power for different ultrasound durations (abscissa). B shows the ratio of left and right chromosomes within chromosomes and between chromosomes (ordinate) obtained using non-contact ultrasound for 4 minutes, 6 minutes, and 10 minutes (abscissa).

[0054] FIG13 shows the proportion of clusters of different sizes (ordinate) obtained by using non-contact ultrasound for 4 min, 6 min, and 10 min (abscissa).

[0055] Figure 14 shows the single-stranded primer adapter sequences used to generate odd-numbered round primers and even-numbered round primers for labeling NHS magnetic beads. The 5'P modification indicates a primer with a phosphorylation modification at the 5' end. The nucleotide sequences of primers oddBo1-oddBo100 correspond to sequences 1-100 in the sequence listing; the nucleotide sequences of primers oddTop1-oddTop100 correspond to sequences 101-200 in the sequence listing; the nucleotide sequences of primers evenBo1-evenBo100 correspond to sequences 201-300 in the sequence listing; and the nucleotide sequences of primers evenTop1-evenTop100 correspond to sequences 301-400 in the sequence listing. In sequences 1-400 in the sequence listing, the first nucleotides are all phosphorylated: the first nucleotide G in sequences 1-100 represents deoxyguanosine 5′-monophosphate (dGMP); the first nucleotide C in sequences 101-200 represents deoxycytidine 5′-monophosphate (dCMP); the first nucleotide T in sequences 201-300 represents deoxythymidine 5′-monophosphate (dTMP); and the first nucleotide A in sequences 301-400 represents deoxyadenosine 5′-monophosphate (dAMP).

[0056] Figure 15 shows the single-stranded primer adapter sequences for the X-direction double-stranded primer and the Y-direction double-stranded primer used to generate spatial barcode labels. The 5'P modification represents a primer with a phosphorylation modification at the 5' end; TAMRA (Tamra) represents a primer with a TAMRA fluorescent modifier, and TAMRAdT (tamradT) represents a T base with a TAMRA fluorescent modifier; FAM represents a primer with a FAM fluorescent modifier, and FAMdT (famdT) represents a T base with a FAM fluorescent modifier; biotin represents a primer with a biotin fluorescent modifier, and biotindT represents a T base with a biotin fluorescent modifier. The nucleotide sequences of primers oddtamBo1-oddtamBo100 correspond to sequences 401-500 in the sequence listing; the nucleotide sequences of primers evenfamBo1-evenfamBo100 correspond to sequences 501-600 in the sequence listing; and the nucleotide sequences of primers evenbioTop1-evenbioTop100 correspond to sequences 601-700 in the sequence listing. In sequences 401-700 in the sequence listing, the first nucleotides are all phosphorylated: the first nucleotide G in sequences 401-500 represents deoxyguanosine 5′-monophosphate (dGMP); the first nucleotide T in sequences 501-600 represents deoxythymidine 5′-monophosphate (dTMP); the first nucleotide A in sequences 601-700 represents deoxyadenosine 5′-monophosphate (dAMP). At the same time, " / tamradT / " in sequence 401-500 represents thymidine deoxynucleotide modified with 6-TAMRA (6-carboxytetramethylrhodamine); " / famdT / " in sequence 501-600 represents thymidine deoxynucleotide modified with 5-FAM (5-carboxyfluorescein); and " / biotindT / " in sequence 601-700 represents thymidine deoxynucleotide modified with biotin. Modes for Carrying Out the Invention

[0057] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0058] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0059] The compositions of the experimental reagents used in the embodiments of the present invention are as follows:

[0060] DSG stock solution: 50 mg of DSG powder was dissolved in 306 μl of DMSO.

[0061] Protease inhibitor stock solution: dissolve 1 Protease Inhibitor Cocktail tablet in 500 μl DMSO.

[0062] Permeabilization buffer 1: 50 mM HEPES (pH 7.5), 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 140 mM NaCl (pH 7.5), 0.25% Triton X-100, 0.5% NP-40, 10% glycerol, and the rest is ultrapure water.

[0063] Permeabilization buffer 2: 50 mM HEPES (pH 7.5), 1.5 mM EDTA (pH 8.0), 1.5 mM EGTA (pH 8.0), 200 mM NaCl (pH 7.5), and the rest is ultrapure water.

[0064] Permeabilization buffer 3: 50 mM HEPES (pH 7.5), 1.5 mM EDTA (pH 8.0), 1.5 mM EGTA (pH 8.0), 100 mM NaCl (pH 7.5), 0.5% sodium N-lauroylsarcosinate, 0.5% sodium deoxycholate, and the remainder is ultrapure water.

[0065] 10× annealing buffer: 100 mM Tris (pH 7.5), 1.5 mM EDTA (pH 8.0), 1.5 mM EGTA (pH 8.0), 2 M LiCl, and the rest is ultrapure water.

[0066] Channel termination buffer: 0.1% Triton X-100, 1.5 mM EDTA (pH 8.0), 1.5 mM EGTA (pH 8.0) dissolved in DPBS.

[0067] Channel wash buffer: 0.1% TritonX-100 dissolved in DPBS.

[0068] Magnetic bead coupling buffer: 0.1% SDS dissolved in PBS.

[0069] Magnetic bead blocking buffer: 0.5% Triton X-100, 0.5 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) dissolved in Tris-HCl (pH 7.5).

[0070] Magnetic bead washing buffer: 20 mM Tris-HCl (pH 7.5), 50 mM EDTA (pH 8.0), 50 mM EGTA (pH 8.0), 50 mM NaCl, 0.2% Triton X-100, 0.2% NP-40, 0.2% sodium deoxycholate, and the rest is ultrapure water.

[0071] Magnetic bead termination buffer: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.2% Triton X-100, 0.2% NP-40, 0.2% sodium deoxycholate, and the rest is ultrapure water.

[0072] Proteinase K buffer: 20 mM Tris-HCl (pH 7.5), 10 mM EDTA (pH 8.0), 50 mM NaCl, 2% sodium N-lauroylsarcosinate, and the remainder is ultrapure water.

[0073] 1× B&W buffer with Tween-20: 5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA (pH 8.0), 0.05% Tween-20, and the remainder is ultrapure water.

[0074] 2×B&W buffer: 10mM Tris-HCl (pH7.5), 2M NaCl, 1mM EDTA (pH8.0).

[0075] Tris & Tween buffer: 10 mM Tris-HCl (pH 7.5), 0.1% Tween-20 dissolved, and the rest ultrapure water.

[0076] The sources of the reagents used in the examples of the present invention are as follows:

[0077] Chemical reagents and buffers: DSG cross-linker (Thermo Fisher Scientific, Cat. No. 20593), DPBS buffer (Thermo Fisher Scientific, Cat. No. 14190144), HEPES buffer (pH = 7.4) (Sigma-Aldrich, Cat. No. H0887-100ML), glycine (Sigma-Aldrich, Cat. No. 50046), EDTA-free protease inhibitor cocktail (Roche, Cat. No. 11697498001), N-lauroylsarcosine sodium salt solution (Sigma-Aldrich, Cat. No. L7414), sodium deoxycholate (Sigma-Aldrich, Cat. No. D6750), NP-40 (Sigma-Aldrich, Cat. No. NP40S-500ML), Triton X-100 (Yisheng, Cat.No.20107ES20), SDS (Sigma-Aldrich, Cat.No.D6750), 0.5M EGTA buffer (pH=8.0) (Biyuntian, Cat.No.SL3071-100ml), 0.5M EDTA buffer (pH=8.0) (Biyuntian, Cat.No.ST063-500ml), 5M sodium chloride solution (Biyuntian, Cat.No.ST348-500ml), 100mM PMSF (Biyuntian, Cat.No.ST506), enzyme-free ultrapure water (Biyuntian, Cat.No.ST876-500ml), 16% formaldehyde solution (Pierce, Cat.No.28906).

[0078] Molecular biology reagents: HinP1I endonuclease (NEB, Cat. No. R0124S), MseI endonuclease (NEB, Cat. No. R0525M), proteinase K (Thermo Fisher, Cat. No. EO0491), T4 DNA ligase (Norwegian, Cat. No. N103-01), 100 mM ATP solution (Norwegian, Cat. No. DD4106-PA-01), Kelnow large fragment without exonuclease activity (Enzymatics, Cat. No. P7010-HC-L), KAPA amplification enzyme premix (KAPABiosystems, Cat. No. KK2602), 10 mM dNTPs (Thermo Fisher Scientific, R0192).

[0079] Magnetic beads and kits: NHS magnetic beads (Smart-Lifebiosciences, Cat. No. SM030050), DNA sorting magnetic beads (Norvozymes, N411-01), streptavidin magnetic beads (Thermo Fisher Scientific, Cat. No. R0192), Qubit fluorescence quantitative kit (Yisheng, Cat. No. 12640ES76), gel extraction kit (Norvozymes, Cat. No. DC301-01). The primer sequences ordered in the examples of the present invention are shown in Table 1 below:

[0080] Table 1. DNA adapter primer sequences Note: The 5'P modification indicates a phosphorylation modification at the 5'-terminal nucleotide of the primer sequence. The first nucleotide T in sequence 703 represents deoxythymidine 5'-monophosphate (dTMP); the first nucleotide G in sequence 704 represents deoxyguanosine 5'-monophosphate (dGMP); and the first nucleotide G in sequence 713 represents deoxyguanosine 5'-monophosphate (dGMP). TAMRAdT represents a T base with a TAMRA fluorescent moiety; FAMdT represents a T base with a FAM fluorescent moiety; biotindT represents a T base with a biotin fluorescent moiety; and N represents any of A, T, C, or G.

[0081] The remaining double-stranded primer sequences are shown in Figures 14 and 15. Since there are two types of microfluidic chips, one with 96 wells and the other with 100 wells, the table lists the primers used for the 100-well chip. When using a 96-well chip, only the first 96 sequences need to be taken.

[0082] The final primers used in the examples of the present invention are as follows:

[0083] The following primers were mixed in equal moles and annealed to obtain the primers required for the final experiment:

[0084] Anneal the DNA adapter top (Mse I) in Table 1 with the DNA adapter bot in Table 1 to generate the Mse I-DNA adapter primers at a final concentration of 10 μM. The reaction conditions were as follows: add 1× annealing buffer to each of the two single-stranded adapter primers, mix them in equimolar amounts, heat at 95°C for 3 minutes, and then anneal at a rate of 0.1°C / s to generate the double-stranded DNA adapter primers.

[0085] The DNA adapter top (Hinp1I) in Table 1 was annealed with the DNA adapter bot in Table 1 to generate a Hinp1I-DNA adapter primer at a final concentration of 10 μM. The reaction conditions were as follows: 1× annealing buffer was added to each of the two single-stranded adapter primers, mixed in equimolar amounts, and heated at 95°C for 3 minutes before annealing at a rate of 0.1°C / s to generate a double-stranded DNA adapter primer.

[0086] According to the single-stranded primer adapter sequences in Figures 14 and 15, the following double-stranded primers were annealed respectively:

[0087] Anneal the single-stranded primers oddTopα (where α = 1.2...100) and oddBoβ (β = 1.2...100) in Figure 14 to obtain 100 odd double-stranded primers (Odd1-Odd100, i.e., in Figure 14 , oddTop1 and oddBo1 anneal to obtain the odd double-stranded primer Odd1, oddTop2 and oddBo2 anneal to obtain the odd double-stranded primer Odd2, ..., and oddTop100 and oddBo100 anneal to obtain the odd double-stranded primer Odd100). The final concentration is 5 μM. The primers are dispensed into a multiwell plate, 3 μL per well. These 100 odd double-stranded primers are used as odd-numbered round primers for labeling NHS magnetic beads.

[0088] The single-stranded primers evenTopα (α = 1.2...100) and evenBoβ (β = 1.2...100) in Figure 14 were annealed separately to obtain 100 even double-stranded primers (Even1-Even100; i.e., in Figure 14 , evenTop1 and evenBo1 were annealed to obtain the even double-stranded primer Even1; evenTop2 and evenBo2 were annealed to obtain the even double-stranded primer Even2; ..., evenTop100 and evenBo100 were annealed to obtain the even double-stranded primer Even100). The final concentration was 5 μM. The primers were dispensed into a multi-well plate, with 3 μL dispensed per well. These 100 even double-stranded primers were used as even-numbered round primers for the NHS magnetic beads.

[0089] The single-stranded primer oddTopα (α=1.2...100) in Figure 14 and the single-stranded primer oddtamBoβ (β=1.2...100) in Figure 15 were annealed respectively to obtain 100 odd double-stranded fluorescent primers (oddtam1-oddtam100, i.e., oddTop1 in Figure 14 and oddtamBo1 in Figure 15 were annealed to obtain the oddtam double-stranded primer oddtam1, oddTop2 in Figure 14 and oddtamBo2 in Figure 15 were annealed to obtain the oddtam double-stranded primer oddtam2, ..., oddTop100 in Figure 14 and oddtamBo100 in Figure 15 were annealed to obtain the oddtam double-stranded primer oddtam100), with a final concentration of 25 μM. The primers were dispensed into a multi-well plate, with X μL allocated to each well (X is the number of samples this time). These 100 odd double-stranded fluorescent primers are X-direction primers labeled with spatial barcodes, X-barcodes.

[0090] Anneal the single-stranded primers evenbioTopα (α = 1.2...100) and evenfamBoβ (j = 1.2...100) in Figure 15 to obtain 100 even double-stranded fluorescent primers (evenFAM1-evenFAM100, i.e., in Figure 15 , evenbioTop1 and evenfamBo1 anneal to obtain the evenfam double-stranded primer evenfam1, evenbioTop2 and evenfamBo2 anneal to obtain the evenfam double-stranded primer evenfam2, ..., and evenbioTop100 and evenfamBo100 anneal to obtain the evenfam double-stranded primer Evenfam100). The final concentration is 25 μM. Dispense the primers in a multiwell plate, dispensing X μl per well (X is the number of samples). These 100 even double-stranded fluorescent primers serve as the Y-barcode primers for spatial barcode labeling. EvenbioTopα single-stranded primers are labeled with biotin, so Y-barcode double-stranded primers are modified with biotin.

[0091] Ter-eve-top-iα (α = 1.2.3.4) and Ter-oe-botiβ (β = 1.2.3.4) were annealed separately to obtain four terminal library construction primers (Ter1-Ter4) with a final concentration of 25 μM. That is, Ter-eve-top-i1 in Table 1 was annealed with Ter-oe-boti1 in Table 1 to obtain terminal library construction primer Ter1, Ter-eve-top-i2 in Table 1 was annealed with Ter-oe-boti2 in Table 1 to obtain terminal library construction primer Ter2, Ter-eve-top-i3 in Table 1 was annealed with Ter-oe-boti3 in Table 1 to obtain terminal library construction primer Ter3, and Ter-eve-top-i4 in Table 1 was annealed with Ter-oe-boti4 in Table 1 to obtain terminal library construction primer Ter4.

[0092] The reaction conditions for generating double-stranded primers from the above two single-stranded primer adapter sequences are as follows: 1× annealing buffer is added to the two single-stranded adapter primer species respectively, mixed in equimolar amounts, heated at 95°C for 3 minutes, and then annealed at a rate of 0.1°C / s to finally obtain DNA double-stranded adapter primers.

[0093] The mouse breeding and breeding process used in the embodiment of the present invention is as follows:

[0094] The breeding process involved placing 3-4 commercially available mice (Vitamin Liva, approximately 8 weeks old) in standard ventilated cages under a 12-hour light / 12-hour dark cycle. The ambient temperature was 25°C and the relative humidity was 50%. For mating, the male and female mice were caged in a 1:1 ratio at 10 pm and the presence of vaginal plugs in the female mice was checked at 7 am the following morning. Pregnant mice were housed individually and recorded as 0.5 days pregnant. Weights were recorded daily thereafter.

[0095] The mouse embryo isolation process used in the embodiment of the present invention is as follows:

[0096] After anesthesia, the mouse was sacrificed by dislocating the neck. The abdomen was disinfected with medical alcohol. An incision was made along the midline of the abdomen near the diaphragm, and the uterus was removed and placed in a DPBS solution. The uterus was carefully opened, the embryos separated, and placed in a new tube. The embryos were washed several times with DPBS until the wash solution no longer turned red.

[0097] The PDMS chip used in the embodiment of the present invention is 0.5 cm×0.5 cm, with a resolution of 25 μm (upper figure in FIG1 ) and 50 μm (lower figure in FIG1 ), and has 96 channels in the horizontal and vertical directions, respectively ( FIG1 ).

[0098] Example 1: Establishment of a method for spatial three-dimensional genomic sequencing based on a microfluidic chip

[0099] 1. Cross-linking to fix the conformation of nucleic acids and proteins

[0100] A chip reaction pool was cast using polydimethylsiloxane (PDMS) (ordered from Shanghai Pengzan Biotechnology Co., Ltd.). The following processes after the second cross-linking step were performed within the PDMS chip reaction pool (sample reaction area A in Figure 1):

[0101] (1) Fresh embryos of CD-1 mice (Wei Tonglihua, SPF grade) were placed in an embedding box and embedded with OCT. The embedded tissue blocks were sliced ​​using a freezing microtome with a step size of 10 μm.

[0102] (2) Remove excess OCT embedding medium from frozen tissue sections using ultrapure water and blow dry in a clean bench.

[0103] (3) The PDMS reaction pool is attached to the tissue slice and fixed with a clamp. The central reaction area (sample area) is the area where the tissue slice area and the PDMS reaction pool are attached (shown as the central reaction area in Figure 1). The PDMS chip in Figure 1 includes a sample loading area (to the left of the central reaction well area) and a sample discharge area (to the right of the central reaction area) with 96 sample inlets. From the start of crosslinking to the end of DNA linker ligation, the reaction is carried out in the reaction pool.

[0104] (4) Add a DSG cross-linking system containing 0.8 μl of DSG mother solution and 1199.2 μl of DPBS solution into the PDMS reaction pool, cross-link at room temperature for 45 min and shake gently.

[0105] (5) Use a pipette to aspirate and discard the waste liquid from the reaction pool, and add a formaldehyde cross-linking system containing 50 μl of 16% formaldehyde solution and 150 μl of DPBS solution and incubate at room temperature for 10 minutes.

[0106] (6) Use a vacuum pump to suck out and discard the waste liquid from the reaction pool, add 400 mM glycine (dissolved in DPBS) to the PDMS reaction pool to terminate the system and gently shake at room temperature for 5 minutes to terminate the reaction.

[0107] 2. Permeabilization

[0108] Permeabilize the cells to permeabilize the cell and nuclear membranes, allowing the reagents to fully enter the nucleus for reaction. Specifically, aspirate and discard the waste liquid. Add permeabilization working solutions 1, 2, and 3 (with 1% protease inhibitor stock solution added to each) to the PDMS reaction cell, and incubate at 4°C for 10 minutes each.

[0109] 3. DNA adapter ligation

[0110] (1) Use a vacuum pump to suck and discard the waste liquid from the reaction pool, add SDS system (dissolved in ultrapure water) with a final concentration of 0.3% to the PDMS reaction pool (to bind to the protein and cause it to deform and precipitate), and react at 65°C for 10 minutes.

[0111] (2) Aspirate and discard the waste liquid, add TritonX-100 termination system to the PDMS reaction pool and react at 37°C for 10 minutes.

[0112] (3) Use a vacuum pump to suck and discard the waste liquid from the reaction pool, add a double enzyme digestion and ligation system to the PDMS reaction pool (add 10 μl T4 DNA ligase, 10 μl MseI, 10 μl HinP1I, 20 μl 10×rCUT smart buffer, 5 μl Triton X-100, 20 μl 50% PEG8000, 1 μM DNA adapter ligation primer (Mse I) and 1 μM DNA adapter ligation primer (Hinp1I), 10 μl ATP per 200 μl system) for double enzyme digestion and DNA adapter (Mse I-DNA adapter ligation primer and Hinp1I-DNA adapter ligation primer dissolved in ultrapure water) ligation, react at 37°C for 3 hours, and obtain a tissue slice with added nucleic acid adapters. Separate the tissue slice with added nucleic acid adapters from the PDMS chip to obtain a tissue slice with added nucleic acid adapters after separation.

[0113] 4. Overall spatial position marking - spatial barcode connection and positioning of spatial position (this process needs to be carried out within the PDMS microfluidic chip, taking a chip with a resolution of 25μm and a total of 96 injection holes as an example):

[0114] The PDMS microfluidic chip includes a sample loading area, a central area, and a sample output area (B and C in Figure 1 are the microfluidic chips in the X and Y directions, respectively). After the tissue slices with nucleic acid adapters added after separation are assembled with the central area of ​​the PDMS microfluidic chip (B and C in Figure 1), the nucleic acids on the slices are labeled with barcode fluorescent primers with different numbers in the X and Y directions of the tissue slices (X1-X 96 , Y1-Y are marked on the Y axis 96 ) to perform two rounds of barcode labeling on tissue slices in the vertical direction of space. The reaction is carried out in the central area of ​​the PDMS microfluidic chip (the central area of ​​B or C in Figure 1). The tissue slices are connected in situ at the intersection through the two sets of barcode systems to form an integration point of tissue pixels. The 96-well PDMS microfluidic chip can divide the tissue slice plane into 96×96 (i×j) pixels, each of which contains a different combination of barcodes X and Y (X i Y j, i is any natural number from 1 to 96, j is any natural number from 1 to 96), forming a two-dimensional image, correlating tissue morphology with spatial omics, and achieving spatial position marking at the micron level. The barcode fluorescent primer X-barcod (X1-X 96 With TAMRA fluorescent group) and barcode fluorescent primer Y-barcode (Y1-Y 96 The X-barcode (with a FAM fluorescent group) has different fluorescent groups, which produce different colors of fluorescence under the action of excitation light (X-barcode produces red fluorescence, Y-barcode produces green fluorescence). At the same time, the Y-barcode double-stranded primer is modified with biotin.

[0115] The specific steps are as follows:

[0116] (1) Dispense the Odd double-stranded fluorescent primer into a 96-well plate in advance, 1 μl per well.

[0117] (2) Clean the slides with ultrapure water and place them on a clean bench to dry. Prepare 200 μl of ligation mixture (12.5 μl of T4 DNA ligase (rapid), 100 μl of 2×T4 DNA ligase rapid buffer, and a final concentration of 0.5% Triton X-100 dissolved in ultrapure water).

[0118] (3) 4 μl of the ligation mixture was mixed with each primer in a 96-well plate to a total volume of 5 μl.

[0119] (4) Use wafer film to clean the surface of the microfluidic chip and the glass slide, fix them with a clamp, and install the drainage device.

[0120] (5) Samples were loaded into the 96-well sample loading channel of the microfluidic chip (the sample loading area of ​​B in FIG1 ). After the sample loading was completed, a vacuum pump was used to aspirate the sample at the sample outlet (the sample outlet area of ​​B in FIG1 ), so that the liquid slowly flowed through the central area (the central area of ​​B in FIG1 ), and the reaction was allowed to stand at room temperature for 10 min.

[0121] (6) After the reaction is completed, channel cleaning buffer is added to the 96-well sample channel of the PDMS microfluidic chip for cleaning.

[0122] (7) After the liquid is completely drained, a fluorescence microscope is used to detect and photograph the tissue sections, and a fluorescence detection image is obtained after the spatial barcode fluorescent barcode primer is marked in the X-axis direction (the left image in the upper figure and the left image in the lower figure in Figure 2).

[0123] (8) Repeat the process (1)-(5) to connect the Y-axis spatial barcode fluorescent barcode primer (Y1-Y 96 ).

[0124] (9) After the ligation reaction is completed, the channel is washed with a channel termination buffer and photographed using a fluorescence microscope to obtain fluorescence detection images of the tissue section after the spatial barcode fluorescent barcode primer is marked in the Y-axis direction (the right image of the upper figure and the right image of the lower figure in Figure 2). The results show that the fluorescence detection image after the spatial barcode fluorescent barcode primer is marked in the X-axis direction shows fluorescence, and the fluorescence detection image after the spatial barcode fluorescent barcode primer is marked in the Y-axis direction shows fluorescence, so the spatial barcode in both the X-axis and Y-axis directions is successfully marked.

[0125] 5. Ultrasonic fragmentation of tissue sections - partial lysis:

[0126] The tissue sections are ultrasonically disrupted for partial lysis (lysis only to the level of nano-scale macromolecular complexes) to obtain lysis products containing different protein-nucleic acid complexes. The nucleic acids on the different protein-nucleic acid complexes all carry their spatial position barcodes on the tissue sections.

[0127] The tissue sections on the slides were scraped off using a double-sided soft blade and dissolved in 100-150 μl of permeabilization buffer 3, and then subjected to non-contact ultrasound (instrument model PLUS, select high power, total duration is 10 minutes, 10 seconds on and 10 seconds off alternately).

[0128] 6.NHS conjugation

[0129] NHS-activated magnetic beads were used for NHS coupling reaction in magnetic bead coupling buffer to couple the macromolecular complexes in the cleavage product obtained in step 5 to the NHS-activated magnetic beads. The steps are as follows:

[0130] (1) Take 1 mL of NHS magnetic beads for each sample, place them on a magnetic rack to remove the storage solution and wash them with 600 μL of pre-cooled 1 mM hydrochloric acid and 600 μL of pre-cooled PBS buffer in sequence. Then resuspend them with 1 mL of coupling buffer and add them to the ultrasonic lysate obtained in step 5. Then, place them on a rotary shaker and incubate them at 4°C overnight for coupling reaction.

[0131] (2) After the NHS coupling reaction was carried out overnight, the NHS magnetic beads were terminated with 600 μl of magnetic bead blocking buffer and blocked in a shaker at 4°C, with the liquid changed every 0.5 h.

[0132] (3) The beads were washed with pre-cooled magnetic bead washing buffer by rotation for 5 min each time, for a total of four times to obtain NHS magnetic bead-coupled samples.

[0133] 7. Four rounds of barcode ligation of NHS magnetic beads - partial labeling:

[0134] Using the SPLiT-seq (split-pool ligation-based transcriptome sequencing) method, DNA from NHS magnetic bead-coupled samples (macromolecular complexes coupled to NHS magnetic beads) was subjected to four rounds of barcoding (50 μm) or three rounds of barcoding (25 μm) via four (50 μm resolution) or three (25 μm resolution) split and pooling steps, respectively. Different protein-nucleic acid complexes were labeled with different combinations of barcodes (each combination contained four different barcodes in series (50 μm resolution, represented by the 4× split barcode in Figure 3) or three different barcodes in series (25 μm resolution)) to distinguish different protein-nucleic acid complexes. Nucleic acids within the same protein-nucleic acid complex were labeled with the same combination of barcodes.

[0135] The principle of the four-round "split-pool" step in SPLiT-seq technology is as follows:

[0136] 1) In the first round of reaction, all complexes coupled to NHS-activated magnetic beads are dispersed into reaction system 1 containing k different barcode tags, and the barcode tags from the first round are added to obtain complexes with k different barcode tags added thereto. Then, all the complexes with k different barcode tags added thereto are mixed to obtain an n1 mixture; the nucleic acid fragments in the n1 mixture carry k different barcode tags;

[0137] 2) In the second round of reaction, all the complexes with k different barcode tags added in the n1 mixture are dispersed again into the reaction system 2 containing k different barcode tags, and k different barcode tags are added in the second round to obtain complexes with k different barcode tags added again, and then the complexes with k different barcode tags added again are mixed to obtain the n2 mixture; the nucleic acid fragments in the n2 mixture have k 2 Different barcode labels;

[0138] 3) In the nth round of reaction, the n (n-1)All the complexes with (n-1) rounds of k different barcode labels added to the mixture are dispersed into the reaction system n containing k different barcode labels. The k different barcode labels in the nth round are added to obtain the complexes with k different barcode labels added in the nth round. Then, the complexes with k different barcode labels added in the nth round are mixed to obtain n (n) mixture; n (n) The nucleic acid fragments in the mixture carry k n Different barcode labels;

[0139] n is a natural number greater than 1 and less than or equal to 5, and k is a natural number greater than 1 and less than or equal to 100.

[0140] The specific steps are as follows:

[0141] (1) The 96 odd double-stranded primers (Odd1-Odd96) obtained by annealing the single-stranded primers in Figure 14 were pre-packed into a 96-well plate, and 200 μl of NHS magnetic bead ligation system (5 μl T4 DNA ligase (rapid), 100 μl 2×T4 DNA ligase rapid buffer, final concentration 0.05% Triton X-100, 0.05% NP-40, 0.05% sodium deoxycholate dissolved in ultrapure water) were prepared.

[0142] (2) Each NHS magnetic bead-coupled sample was resuspended using 1 mL of the ligation system and dispensed into a 96-well plate containing an Odd double-stranded primer using a reagent tank.

[0143] (3) Seal the 96-well plate and perform mixing reaction on an Eppendorf ThermoMixer at 30°C for 1 h.

[0144] (4) Then, 60 μl of magnetic bead termination buffer was added to each well and the reaction was stopped after standing for 15 min. The samples from all wells were collected and placed in the same total pool.

[0145] (5) Transfer the solution to a 15 mL conical tube and place it on a magnetic stand. Remove most of the solution, leaving 0.5 mL. Transfer it to a 1.5 mL EP tube and remove the solution again. Wash the beads three times with 600 μl of magnetic bead washing buffer, each time for at least 5 minutes.

[0146] (6) Repeat the process (1) to (5) once to perform the second round of barcode addition, adding 96 even double-stranded primers (Even1-Even96) obtained by annealing the single-stranded primers in Figure 14.

[0147] (7) Repeat the process (1) to (5) again to perform the third round of barcode addition, and once again add 96 types of odd double-stranded primers (Odd1-Odd96) obtained by annealing the single-stranded primers in Figure 14.

[0148] (8) Repeat the process (1)-(5) again to perform the fourth round of barcode addition, and once again add 96 even double-stranded primers (Even1-Even96) obtained by annealing the single-stranded primers in Figure 14.

[0149] (9) Connect the terminal library primer (one of Ter1-Ter4) to the read2 sequencing adapter. The system for each 200 μl is as follows: 5 μl T4 DNA ligase (rapid), 100 μl 2×T4 DNA ligase rapid buffer, final concentration of 1 mM terminal library primer (Ter1-Ter4), and the rest is supplemented with ultrapure water.

[0150] 8. Overall cleavage - anti-crosslinking process:

[0151] The protein-nucleic acid complexes with different barcode combinations (X-barcode-Y-barcode-Odd-Even-Odd-Even) obtained in step 7 are reverse-crosslinked to obtain de-crosslinked, non-visualized nucleic acid fragments. The de-crosslinked nucleic acid fragments carry chromatin conformation information derived from a tandem combination of two spatial position information (XiYj) labels (X-barcode-Y-barcode) and four barcodes (split1-split4: Odd-Even-Odd-Even).

[0152] The specific steps are as follows:

[0153] (1) Reverse cross-link the NLS beads in proteinase K buffer. Add 430 μl proteinase K buffer, 30 μl 5M NaCl, and 40 μl Proteinase K to every 500 μl system.

[0154] (2) Place the reverse cross-linked tube at 55°C and shake overnight for 12 hours.

[0155] (3) After overnight reverse cross-linking, 1% 100 mM PMSF was added to the solution and the proteinase K was inactivated by rotation at room temperature for 20 minutes to obtain a reverse cross-linking mixture.

[0156] 9. Biotin Purification

[0157] Since the Even double-stranded fluorescent protein is modified with biotin in step 4, the reverse cross-linking mixture is purified using magnetic beads coated with streptavidin C1 to purify the nucleic acid (DNA) fragments obtained in step 8.

[0158] ① For each sample, take 40 μl of streptavidin-modified magnetic beads and wash them three times with 800 μl of 1× B&W buffer for 5 minutes. Finally, resuspend them in 100 μl of 2× B&W buffer to obtain a streptavidin magnetic bead suspension.

[0159] ② Add the anti-crosslinked mixed solution obtained in step 8 to the streptavidin magnetic bead suspension and shake at room temperature at 30 rpm for 1 h;

[0160] ③. Wash twice with 400 μl 1× B&W buffer, 5 min each time

[0161] ④. Resuspend in 400 μl 10 mM Tris (0.1% Tween-20) to obtain a target nucleic acid fragment mixture.

[0162] 10. Library Construction and Sequencing

[0163] (1) A single-end random amplification system containing 6 random bases at the 3' end was used to construct the library, and a read1 sequencing adapter was introduced: a single-stranded random primer containing 6 random bases N (N is any of A, T, C or G) at the 3' end and a sequencing adapter sequence at the 5' end was randomly combined with the target nucleic acid fragment by annealing and renaturing to obtain a renatured product. The renatured product was then completely amplified using DNA polymerase, and a sequencing library containing two sequencing adapter sequences and a fragment length suitable for the machine (350-750 bp) was obtained by fragment sorting.

[0164] The specific steps are as follows: resuspend the magnetic beads containing the target DNA fragment in the target nucleic acid fragment mixture in 50μl of single-end random amplification system (5μl KLENOW blue buffer, 2μl dNTPs, 10μl single-stranded amplification primer, and the rest is made up of ultrapure water) and place at 95°C for 45 seconds, then quickly chill on ice. Then, add 1μl of Klenow enzyme (Klenow fragment of DNA polymerase with strand displacement activity) and increase the temperature at a rate of 0.1°C / s until the final temperature reaches 37°C. Then, incubate for 30 minutes to obtain the incubation product.

[0165] After the magnetic beads in the incubation product were adsorbed using a magnetic stand, the supernatant was added to 50 μl of library amplification system (25 μl KAPA2×MIX, 2 μl PCR1, 2 μl PCR2 (i1-i4), and the rest was supplemented with ultrapure water) for amplification (a total of 15 cycles) to obtain the amplified product library:

[0166] The amplified product library was subjected to gel recovery fragment screening, and bands between 350-750 bp were selected.

[0167] Using a Qubit and fragment analyzer for quality control, the final library should be at least 50 ng in total, with fragment lengths ranging from 350 to 750, to produce a sequencing library. Figure 4 shows the DNA fragment distribution range for qualified samples.

[0168] The sequencing library is sequenced using the BGI DNBSEQ-T7 sequencer to obtain sequencing data, which is then analyzed. The spatial position of the DNA fragment on the tissue section is determined based on the spatial position information on the DNA fragment obtained in step 8; the tandem combination of the four barcodes (split1-split4) on the DNA fragment obtained in step 8 is used to determine which DNA fragments have the same barcode combination. DNA fragments with the same barcode combination are spatially close to each other, and ultimately the three-dimensional genomic conformation of chromatin in the tissue section can be obtained.

[0169] 11. Data Analysis

[0170] 11.1 Analysis of Sequencing Library Base Balance and Barcode Ligation Efficiency in Each Round

[0171] The sequencing library balance results are included in the sequencing data file. Since each round contains a fixed 7-base sequence, there will be 7-base imbalance sites between each round. The connection efficiency analysis is to count the proportion of reads containing different numbers of barcode sequences. The higher the proportion of reads containing six-round barcode sequences (two spatial position information (XiYj) markers and four barcode (split1-split4) markers), the higher the connection efficiency. The upper and lower figures in Figure 5 are the library balance results obtained using pair-end double-end sequencing and the evaluation of the number of labels connected to all reads, respectively. The results show that the connection efficiency of each round is over 90%, and after 6 rounds of connection, the connection efficiency exceeds 50%, which is in line with expectations.

[0172] 11.2 Analyze the number of reads, clusters, and DNA interactions at all sites in the space

[0173] All sequences containing six rounds of barcode information are collected together, and the spatial position is located according to the first two rounds of spatial barcodes to obtain the number of effective reads at each site in the space (left figure in Figure 6). The types of barcodes in the last four rounds (split1-split4) at each site are counted to obtain the number of clusters (middle figure in Figure 6). The number of interactions within each cluster is counted according to the existence of pairwise interactions between sequences. The set of interactions within all clusters of a single spatial site is the number of interactions among all DNA fragments at this spatial site (right figure in Figure 6).

[0174] 11.3 Mapping inter- and intrachromosomal interactions across the entire tissue

[0175] The open-source code (https: / / github.com / GuttmanLab / sprite-pipeline / wiki) was used to map the three-dimensional chromosome-genomic interaction. The left figure in Figure 7 is a heat map of interactions between all chromosomes, and the right figure is a heat map of interactions within a single chromosome. The horizontal and vertical axes are the relative positions of the chromosomes, and the color depth represents the interaction strength.

[0176] 11.4 Sequencing Data Filtering

[0177] Since a huge number of small clusters will be generated during ultrasound, these clusters will randomly bind to the NHS magnetic beads, which will cause clusters originating from different chromosomes to combine together to form false positives. In order to eliminate this influence, the present invention first filtered the data, screened out clusters whose sequences originating from the same chromosome accounted for less than 85% of all chromosome sequences, and performed K-means clustering on the filtered data, with the K value set to 30 and the seed value set to 15. The classification results were then mapped to the UMAP two-dimensional space and embryonic space, and named using the organs or tissues corresponding to the spatial position. The results showed that the 30 clustering results obtained by the method of the present invention were spatially consistent with the 16 organs and tissues, respectively, which indicates that the method established by the present invention is feasible in reproducing the chromatin conformation of animal tissues.

[0178] Example 2: Comparative experiment on optimization of the spatial three-dimensional genomics sequencing method of the present invention

[0179] 1. Test DNA adapter primers

[0180] The reagent formula used was the same as that of Example 1 except for the DNA single-stranded linker primer. The overall experimental process was different from that of Example 1 in two aspects:

[0181] (1) The single-end random amplification process during the biotin purification and library construction is missing;

[0182] (2) The process of connecting the terminal library primers during NHS coupling is located after biotin purification;

[0183] Experimental results show that a significant proportion of the sequencing results, reaching 92%, are empty reads resulting from the splint primer ligation with the terminal primer. Sequences containing TCCAAG within the white box in the figure are all empty reads. Figure 11 shows that this is due to a failure to clean the terminal double-stranded library primers before library construction, leading to numerous empty reads. This phenomenon can be effectively avoided by moving the terminal library primer ligation process before biotin purification.

[0184] The sequences of DNA adapter primer (DPM) and terminal library primer are as follows:

[0185] DNA single-stranded linker primer (DPM) sequence:

[0186] DPM-botFAM (HinP1I-MseI):

[0187] 5'-TGACT / i6FAMdT / GTCATGTCTTCCGATCT / iBiodT / GGGTGA-3' (in this primer, " / i6FAMdT / " represents thymidine deoxynucleotide (dT) modified with 6-FAM (6-carboxyfluorescein); " / iBiodT / " represents thymidine deoxynucleotide (dT) modified with Biotin), the nucleotide sequence of this primer corresponds to sequence 718.

[0188] DPM-bot (HinP1I-MseI):

[0189] 5′-TGACTTGTCATGTCTTCCGATCT / iBiodT / GGGTGA-3′ (“ / iBiodT / ” in this primer represents biotin-modified thymidine deoxynucleotide (dT)); the nucleotide sequence of this primer corresponds to sequence 719.

[0190] DPM-top (HinP1I): 5'-GCTCACCCAAGATCGGAAGATCGTAGCCATGTCGT*T*C* / i2OMeG / * / i2OMeG / * / i2OMeG / -3' (The nucleotide G at the 5' end of this primer is phosphorylated, i.e., the first nucleotide G represents deoxyguanosine 5'-monophosphate (dGMP). "*" indicates a thiolation modification, i.e., the nucleotides preceding and following "*" are linked by a phosphorothioate group (structural formula is Formula 1), as shown in Formula 2; " / i2OMeG / " represents 2'-methoxy-modified guanine deoxynucleotide (2'-O-methylguanosine). The nucleotide sequence of this primer corresponds to sequence 720.

[0191] DPM-top (MseI): 5'-TATCACCCAAGATCGGAAGATCGTAGCCATGTCGT*T*C* / i2OMeG / * / i2OMeG / * / i2OMeG / -3' (In this primer, "*" indicates a thiolation modification, i.e., the nucleotide preceding and following "*" are linked by a phosphorothioate group (structural formula is Formula 1), and the connection method is as shown in Formula 2; " / i2OMeG / " indicates 2'-methoxy-modified guanine deoxynucleotide (2'-O-methylguanosine). The nucleotide sequence of this primer corresponds to sequence 721.

[0192] DPM-bot (HinP1I-MseI) was dissolved with DPM-top (HinP1I) and DPM-top (HinP1I) using 1× annealing buffer and mixed in equal molar amounts for annealing to form double-stranded primers containing MseI and HinP1I restriction sites, respectively.

[0193] End library construction primer sequence:

[0194] Ter-top-odd(u)-i2: 5′-AGTTGTCAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAGGACGGAATCCCAACTCCTTGGCTCACA-3′ (the nucleotide A at the 5′ end of this primer is phosphorylated, i.e., the first nucleotide A represents deoxyadenosine 5′-monophosphate (dAMP)). The nucleotide sequence of this primer corresponds to sequence 722;

[0195] Ter-bot-odd / even(u)-i2 (sequence 723): 5'-TGTGAGCCAAGGAGTGTGGGATTCCGTCCTTGTCTTCCTAAGACCGCTTGGCCTCCGACT-3';

[0196] The two primers were dissolved in 1× annealing buffer and mixed in equal molar amounts for annealing to prepare the terminal library primers.

[0197] 2. Testing DNA Enzyme Digestion Methods

[0198] The experimental materials were also mouse brain tissue slices. The reagent formula used was the same as that of Example 1 except for the DNA linker primer.

[0199] XSDNasetop: 5'-TCACCCAAGTACGG*A*A*G-3' (the nucleotide T at the 5' end of this primer is phosphorylated, i.e., the first nucleotide T represents deoxythymidine 5'-monophosphate (dTMP); the G at the 3' end of this primer is C3-spacer (Spacer C3) modified (i.e., the 3' hydroxyl group is blocked); wherein "*" represents a thio modification, i.e., the nucleotide before "*" and the nucleotide after "*" are connected by a phosphorothioate group (the structural formula is Formula 1), and the connection method is as shown in Formula 2); the nucleotide sequence of this primer corresponds to sequence 724.

[0200] XSDNasebot: 5′-TGACTTGCTTCCGTACTTGGGTGAT-3′ (the nucleotide T at the 5′ end of this primer is phosphorylated, ie, the first nucleotide T represents deoxythymidine 5′-monophosphate (dTMP)); the nucleotide sequence of this primer corresponds to sequence 725.

[0201] The above two primers were diluted in 1× annealing buffer in equal moles and mixed for annealing to obtain DNAse I adapter-ligated primers.

[0202] The method used in this example is to fragment DNA using DNase I. The specific process includes four steps: enzyme digestion with DNase I, repair, end addition of A, and DNA linker ligation after cross-linking and permeabilization.

[0203] (1) Add DNase I digestion system (Cat. No. AM2238) to the reaction pool and react at 37°C for 20 min.

[0204] (2) Add magnetic bead termination buffer to the reaction pool and wash twice, then add magnetic bead washing buffer and wash again three times.

[0205] (3) Add the end repair system (NEB, product number E6050S) to the reaction pool and react at room temperature for 1 hour.

[0206] (4) Add the End Add A System (NEB, Product No. E6053S) to the reaction pool and react at 37°C for 1 h.

[0207] (5) Add 200 μl of DNA enzyme I adapter ligation system (5 μl of T4 DNA ligase (rapid), 100 μl of 2×T4 DNA ligase rapid buffer, final concentration of 1 mM DNA enzyme I adapter ligation primer, and the rest is supplemented with ultrapure water) to the reaction pool and react at 30°C for 30 min. Then, the spatial barcode ligation process is the same as in Example 1.

[0208] The final fluorescence results of this comparative experiment taken using a fluorescence microscope (right figure in Figure 10) were compared with the fluorescence in the method of the present invention in Example 1 (left figure in Figure 10). The results show that under the same exposure time and intensity conditions, the final ligation efficiency of DNA fragmentation using DNase I in this example is much lower than the ligation efficiency of DNA fragmentation using Mse I and HinP1I for double enzyme digestion in Example 1.

[0209] 3. Modify the biotin modification site to improve the capture efficiency of the fragment

[0210] The experimental materials were also mouse brain tissue slices. The reagent formula used was the same as that of Example 1 except for the second round of space primers. The other reagents and methods were the same as those in Example 1.

[0211] In this step, the modification site of the even primer with barcode number 1 is as follows:

[0212] evenTop1: 5'-AGTTGTCACGTCAGCCGCAGTATC-3' (the nucleotide A at the 5' end of this primer is phosphorylated, i.e., the first nucleotide A represents deoxyadenosine 5'-monophosphate (dAMP)); the nucleotide sequence of this primer corresponds to sequence 726;

[0213] evenBo1: 5′-TGACTTGGATACTGCGGCTGACGT-3′ (the nucleotide T at the 5′ end of this primer is phosphorylated, ie, the first nucleotide T represents deoxythymidine 5′-monophosphate (dTMP)); the nucleotide sequence of this primer corresponds to sequence 727.

[0214] In this step, the example of the DPM primer modification site (adding biotin label) with barcode number 1 is as follows:

[0215] XRBS-2nick-Mse-t (sequence 728): 5′-TATCACCCAAGTACGGAAG-3′;

[0216] XRBS-2nick-Hinp-t (sequence 729): 5′-CGTCACCCAAGTACGGAAG-3′;

[0217] DPM-bot (hinp1-MseI):

[0218] 5′-TGACTTGTCATGTCTTCCGTACT / iBiodT / GGGTGA-3′ (in this primer, “ / iBiodT / ” represents biotin-modified thymidine deoxynucleotide (dT)). The nucleotide sequence of this primer corresponds to sequence 730.

[0219] Theoretically, this experiment involved one round of DPM ligation, two rounds of steric barcode primer ligation, and three rounds of macromolecular complex split-and-pool ligation. Since each round of primers had an identical ligation sequence, the sequencing results should have yielded six highly unbalanced sequences. However, the final sequencing results showed only three highly unbalanced sequences. This is because placing the biotin modification site on the DPM primer allows for biotinylation and capture of DNA from the entire tissue section, masking the unbalanced sites at the linkers in the subsequent three rounds. However, if the biotin modification site is transferred to the even primer, as in Example 1, DNA fragments linked only to the second round of steric barcode primers can be specifically enriched.

[0220] 4. Test contact ultrasound time optimization analysis

[0221] The experimental materials were also the mouse brain tissue slices in Example 1, and also included mouse embryonic slices at E9.5 and E14.5.

[0222] The reagent formula used was the same as in Example 1, and the experimental steps included the cross-linking and permeabilization processes described in Example 1. After permeabilization, the sections were placed in permeabilization buffer 3 using a soft blade and subjected to contact sonication for varying durations using a Fisher FB705220 (ultrasound power of 45 W) with a time gradient of 15 s, 40 s, and 1 min. After sonication, the mixture was mixed thoroughly and 1 μl was pipetted onto a glass slide. After complete drying, DAPI staining solution was added and observation was performed using laser confocal microscopy.

[0223] Figure 9 shows the results of ultrasound at E9.5 for 15s, 40s, and 1min from top to bottom, respectively. Three fields of view were randomly selected for the sample at each ultrasound time.

[0224] This experiment showed that for the three tissue samples, the protein-DNA complex gradually disappeared after 1 minute of contact sonication, indicating that the contact sonication time at this point exceeded the optimal time. However, the size of the protein-DNA complex did not show a relatively stable pattern between 15 seconds and 40 seconds for the three tissues. This may be due to the significant splashing of liquid during the sonication process in the contact sonicator, causing some larger tissue pieces to splash onto the tube wall. It may also be due to the high foaming produced by contact sonication, which affects the sonication effect. Non-contact sonication allows the sample to be broken more evenly in the solution, effectively avoiding the situation where the protein-DNA complex is too different.

[0225] 5. Comparison of different testing times between contact ultrasound and non-contact ultrasound

[0226] The experimental materials were the same as those in Example 1 for embryonic slices at E12.5, and the reagent formulas used were the same as those in Example 1. The experimental steps included all the processes in Example 1. In this example, after permeabilization, the slices were placed in permeabilization buffer 3 using a soft blade and subjected to contact or non-contact ultrasonic treatment for different times (the contact ultrasonic power was 30W and 40W, and the time was 10s, 20s, and 30s; the non-contact ultrasonic treatment was 4min, 6min, and 10min, and the time was 10s, 20s, and 30s respectively; the non-contact ultrasonic treatment was 4min, 6min, and 10min, and the time was 10s, 20s, and 30s respectively). PLUS instrument for the experiment), with a time gradient of 15 s, 40 s, and 1 min.

[0227] The experimental results show that the ratio of intrachromosomal interactions to interchromosomal interactions obtained using contact ultrasound of different intensities does not show a significant trend of change with time extension or power increase (Figure 12A). However, using non-contact ultrasound, it can be found that the ratio of intrachromosomal interactions gradually increases at a time gradient of 4 minutes, 6 minutes, and 10 minutes (Figure 12B). And by counting the number and size of all clusters obtained for each sample, it can be found that as the ultrasound time increases, the proportion of clusters with 2 or more interactions can be obtained. There is a significant increase (Figure 13), which shows that the method of using non-contact ultrasound for tissue fragmentation is highly repeatable and controllable.

[0228] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

[0229] CROSS-REFERENCE TO RELATED APPLICATIONS

[0230] This application claims priority to the Chinese patent application (application number 202410017141.8) filed on January 4, 2024, the entire contents of which are incorporated herein by reference. Industrial Applicability

[0231] The method of the present invention realizes the detection of spatial three-dimensional genome for the first time. Compared with the three-dimensional genome at the tissue level and single-cell level, it can provide spatial position information within the tissue, providing a new technical platform for chromatin biology; compared with previous tissue level and single-cell level single-cell methods, the present invention can detect more interactions between chromosomes, providing technical support for analyzing the contribution of chromosome territory to tissue organ development and cell fate determination; the method of the present invention does not require sophisticated instruments and equipment, the overall process cost is low, and the instrument molds can be reused, which can be used to develop or prepare products for disease detection or products for observing organ development.

Claims

1. A method for detecting the three-dimensional genomic conformation of chromatin, characterized in that: The method includes the following steps: A1) Crosslinking and fixation: After the tissue section to be tested is assembled by fitting with the reaction pool of the PDMS chip, a crosslinking agent is added to the reaction pool for crosslinking to obtain a tissue section to be tested with fixed nucleic acid and protein conformations; A2) Cell permeabilization: The tissue section to be tested is permeabilized in the reaction pool to obtain a permeabilized tissue section to be tested; A3) Nucleic acid linker ligation: The nucleic acid in the permeabilized tissue section to be tested in the reaction pool is double-digested with enzymes and nucleic acid linkers are added to obtain a tissue section with nucleic acid linkers added; the tissue section with nucleic acid linkers added is separated from the PDMS chip to obtain a tissue section with nucleic acid linkers added after separation; A4) Overall spatial position labeling: The tissue section with nucleic acid linkers added after separation is assembled with a microfluidic chip to obtain an assembled microfluidic chip. The microfluidic chip includes a loading port, a central region, and an outlet port. The central region of the assembled microfluidic chip is in contact with the tissue section with nucleic acid linkers added after separation; i kinds of X-labeled mixed solutions from X1 to Xi are added to the injection hole channel and the mixed solutions are sucked into the central region by a vacuum pump to add i kinds of barcode labels to the nucleic acid of the tissue section with nucleic acid linkers added in the X-axis direction in the microfluidic chip to obtain a tissue section labeled in the X-axis direction; Then j kinds of Y-labeled mixed solutions from Y1 to Yj are added to the injection hole channel and the mixed solutions are sucked into the central region by a vacuum pump to add j kinds of barcode labels to the nucleic acid in the tissue section labeled in the X-axis direction in the Y-axis direction in the microfluidic chip to obtain a tissue section with overall spatial position labeling; The X-axis direction is perpendicular to the Y-axis direction; the tissue section marked with the overall spatial position is marked with i×j pixel points, and the nucleic acid in each pixel point contains different combinations of barcodes X and Y, X i Y j ; the X marker and the Y marker carry different fluorescent groups, and the different fluorescent groups produce different colors of fluorescence under the action of excitation light; i is a natural number greater than 1 and less than or equal to the number of sample injection hole channels, and j is a natural number greater than 1 and less than or equal to the number of sample injection hole channels; A5) Ultrasonic fragmentation of the tissue section with overall spatial position labeling: The tissue section with overall spatial position labeling is ultrasonically fragmented to obtain a lysate containing protein-nucleic acid complexes; A6) NHS-activated magnetic bead coupling: The protein-nucleic acid complexes in the lysate are coupled with NHS-activated magnetic beads to obtain a complex coupled with NHS-activated magnetic beads; A7) Partial labeling: The nucleic acid in the complex of the coupled NHS-activated magnetic beads is added with n rounds of k different barcode labels through n rounds of "dispersion - mixing" steps using the SPLiT-seq method, resulting in a complex with k n types of concatenated combined barcodes, and the nucleic acids of different said complexes contain k n types of different said concatenated combined barcodes; the n is a natural number greater than 1 and less than or equal to 5; the k is a natural number greater than 1 and less than or equal to 100; A8) Reverse crosslinking for overall lysis: Reverse crosslink the complex added with the n kinds of tandem combined barcodes to obtain the de-crosslinked nucleic acid fragments, and purify the de-crosslinked nucleic acid fragments to obtain the nucleic acid fragments to be sequenced; the nucleic acid fragments to be sequenced contain the nucleic acid adapter sequence, the n kinds of tandem combined barcode sequences, and the X i Y j barcode sequence; A9) Library construction and sequencing: The nucleic acid fragment to be sequenced is used to construct a sequencing library by binding and amplifying with single-stranded random primers. The sequencing library is sequenced to obtain sequencing data, and the sequencing data is analyzed. Based on the tandem combined barcode sequence and the i Y j barcode sequence, the three-dimensional chromatin spatial genome conformation of the tissue section to be tested is obtained.

2. The method according to claim 1, wherein: The double digestion in A3) is performed using restriction endonucleases Mse I and HinP1I; the nucleic acid linker in A3) includes a linker for the sticky end digested by Mse I and a linker for the sticky end digested by HinP1I.

3. The method according to claim 1, characterized in that: The Y label in A4) has a modification with a biotin group, and the purification in A8) is obtained by adsorbing and purifying by forming a complex between the magnetic bead with streptavidin and the biotin group to obtain the nucleic acid fragment to be sequenced.

4. The method according to claim 1, wherein: The ultrasonic fragmentation in A5) is non-contact ultrasonic fragmentation, and the time of the non-contact ultrasonic fragmentation is greater than or equal to 4 min and less than or equal to 10 min.

5. The method according to claim 1, characterized in that: The single-end random primer binding and amplification includes the following steps: A single-stranded random primer with 6 random bases N at the 3' end and a sequencing linker sequence at the 5' end randomly binds to the nucleic acid fragment to be sequenced by annealing and renaturation to obtain a renatured product, and then the renatured product is completely amplified using DNA polymerase to obtain the sequencing library containing two sequencing linker sequences.

6. The method according to claim 1, wherein: The double digestion described in A3) is double digestion using restriction endonucleases Mse I and HinP1I; the nucleic acid adapter described in A3) includes an adapter for the sticky end digested by Mse I and an adapter for the sticky end digested by HinP1I; the Y label described in A4) is modified with a biotin group, and the purification described in A8) is to obtain the nucleic acid fragment to be sequenced by adsorption purification through the formation of a complex between the magnetic beads with streptavidin and the biotin group.

7. The method according to claim 1, wherein: The double digestion described in A3) is double digestion using restriction endonucleases Mse I and HinP1I; the nucleic acid adapter described in A3) includes an adapter for the sticky end digested by Mse I and an adapter for the sticky end digested by HinP1I; the sonication described in A5) is non-contact sonication, and the time of the non-contact sonication is greater than or equal to 4 min and less than or equal to 10 min.

8. The method according to claim 1, wherein: The double digestion described in A3) is double digestion using restriction endonucleases Mse I and HinP1I; the nucleic acid adapter described in A3) includes an adapter for the sticky end digested by Mse I and an adapter for the sticky end digested by HinP1I; the single-end random primer binding and amplification includes the following steps: a single-stranded random primer with 6 random bases N at the 3'-end and a sequencing adapter sequence at the 5'-end randomly binds to the nucleic acid fragment to be sequenced by annealing and renaturation to obtain a renatured product, and then the renatured product is completely amplified using DNA polymerase to obtain the sequencing library containing two sequencing adapter sequences.

9. The method according to claim 1, wherein: The Y label described in A4) is modified with a biotin group, and the purification described in A8) is to obtain the nucleic acid fragment to be sequenced by adsorption purification through the formation of a complex between the magnetic beads with streptavidin and the biotin group; the sonication described in A5) is non-contact sonication, and the time of the non-contact sonication is greater than or equal to 4 min and less than or equal to 10 min.

10. The method according to claim 1, wherein: The Y label described in A4) is modified with a biotin group, and the purification described in A8) is to obtain the nucleic acid fragment to be sequenced by adsorption purification through the formation of a complex between the magnetic beads with streptavidin and the biotin group; the single-end random primer binding and amplification includes the following steps: a single-stranded random primer with 6 random bases N at the 3'-end and a sequencing adapter sequence at the 5'-end randomly binds to the nucleic acid fragment to be sequenced by annealing and renaturation to obtain a renatured product, and then the renatured product is completely amplified using DNA polymerase to obtain the sequencing library containing two sequencing adapter sequences.

11. The method according to claim 1, wherein: A5) The ultrasonic fragmentation is non-contact ultrasonic fragmentation, and the time of the non-contact ultrasonic fragmentation is greater than or equal to 4 min and less than or equal to 10 min; the single-end random primer binding amplification includes the following steps: a single-stranded random primer with 6 random bases N at the 3'-end and a sequencing adapter sequence at the 5'-end is randomly bound to the nucleic acid fragment to be sequenced by annealing and renaturation to obtain a renatured product, and then the renatured product is completely amplified using DNA polymerase to obtain the sequencing library containing two sequencing adapter sequences.

12. Method for spatial three-dimensional genomics sequencing, characterized in that: The method includes the step of obtaining a spatial three-dimensional genomics sequencing library of a tissue section to be tested using the sequencing library construction method according to any one of claims 1-11, and sequencing the sequencing library.

13. The method for constructing a sequencing library according to any one of claims 1-11.

14. Application of the method according to any one of claims 1-12 in the development or preparation of a product for disease detection.

15. Application of the method according to any one of claims 1-12 in the development or preparation of a product for observing organ development.

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