A method for single-cell nucleus dissociation in tissue sections and its application

Abstract

This invention discloses a method and application for single-cell nucleus dissociation suitable for tissue sections. The method includes: S1, obtaining tissue sections to be processed, attaching them to a carrier surface, and then adding an incubation buffer for incubation; wherein the incubation buffer contains Tris-HCl, NaCl, MgCl2, micro / nano particles, RNase inhibitors, bovine serum albumin, and a nonionic surfactant; S2, transferring the incubated product to a centrifuge tube, gently mixing by pipetting to obtain a nucleus dissociation buffer; S3, filtering and centrifuging the nucleus dissociation buffer, collecting the precipitate, and resuspending it to obtain a nucleus suspension. This single-cell nucleus dissociation method is mild and efficient. The nucleus suspension obtained using this method has high purity of single nuclei and low cytoplasmic debris, and can be directly used for downstream omics analyses such as high-throughput single-cell nuclear sequencing, spatial transcriptomics, and ATAC-seq, demonstrating high application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for the dissociation of single-cell nuclei in tissue sections. Background Technology

[0002] With the development of single-cell nuclear sequencing and space omics technologies, obtaining single-cell nuclear suspensions with high integrity and low fragmentation rate is crucial. Especially for tissue sections and micro-dissected samples with limited sample volume, consistently obtaining high-quality single-cell nuclear suspensions remains a key technological bottleneck.

[0003] Among related technologies, single-cell nucleus dissociation methods mainly include two categories: enzymatic lysis and mechanical grinding. Mechanical grinding primarily achieves cell disruption through physical shearing, resulting in good disruption. However, for small or fragile tissues, it often generates excessive local stress, easily leading to nucleus rupture and increased fragmentation, reducing the purity and usability of the single-cell nucleus suspension. Enzymatic lysis relies on proteases to disrupt the cell membrane. Its lysis mechanism is gentler than mechanical grinding, but due to the relative difficulty in enzyme diffusion and reaction control, it easily leads to nuclear membrane damage and RNA degradation. Furthermore, it is highly sensitive to sample freshness, exhibiting significant limitations. In addition, these methods suffer from poor adaptability to small sample volumes, such as the inability to guarantee consistency and quality of output under small-volume conditions, severely hindering their widespread adoption in high-sensitivity applications such as single-cell nucleus sequencing.

[0004] Therefore, there is an urgent need to develop a gentle, controllable, and adaptable single-cell nucleus dissociation technology to reduce nuclear aggregation, decrease damage and fragmentation, and improve the purity and integrity of single-cell nuclei. Summary of the Invention

[0005] The first objective of this invention is to provide a method for the dissociation of a single cell nucleus.

[0006] The second objective of this invention is to provide the application of the above-described single-cell nucleus dissociation method in the preparation of cell nucleus suspensions.

[0007] A third aspect of the present invention is to provide a detection device.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a method for dissociating a single cell nucleus, comprising the following steps: S1. Obtain the tissue sections to be processed, mount them on the carrier surface, and then add incubation buffer for incubation; The incubation buffer contains Tris-HCl, NaCl, MgCl2, micro / nano particles, RNase inhibitors, bovine serum albumin, and nonionic surfactants. S2. Transfer the incubated product to a centrifuge tube and gently pipette 5-45 times to obtain the initial nucleus dissociation solution. S3. Filter and centrifuge the initial solution of cell nucleus dissociation, collect the precipitate, and resuspend it to obtain a cell nucleus suspension.

[0009] The single-cell nucleus dissociation method according to embodiments of the present invention has at least the following beneficial effects: (1) The single-cell nucleus dissociation method of the present invention is mild and efficient, and is particularly suitable for small-volume tissue sections. Compared with conventional enzymatic lysis (which easily leads to excessive digestion of the nuclear membrane) or strong mechanical grinding (which easily causes nuclear rupture), the method of the present invention achieves mild and controllable release of the cell nucleus through the local mechanical action of micro- and nanoparticles on the cell membrane / cytoskeleton under controlled physical disturbance, which significantly reduces the risk of mechanical damage to the nuclear membrane and improves the dissociation efficiency while maintaining the integrity of the nucleus.

[0010] (2) The cell nucleus suspension obtained by the method of this invention has high purity of single cell nuclei and low cytoplasmic debris. This invention utilizes micro- and nano-particles to help adsorb cytoplasmic debris and reduce cell nucleus aggregation. Combined with low-speed centrifugation, this further significantly reduces the content of debris impurities in the suspension, thereby obtaining a high-quality cell nucleus suspension. The single cell nuclei obtained by the method of this invention can be directly used for subsequent downstream omics analyses such as high-throughput single-cell nucleus sequencing (scRNA-seq), spatial transcriptomics (Visium), and ATAC-seq, without the need for additional purification steps.

[0011] (3) The single-cell nucleus dissociation method of the present invention is simple, time-saving and widely applicable. It can be used for a variety of difficult-to-process sample types (such as liver, heart, lung, brain and tumor), overcoming the limitations of traditional methods on sample volume, freshness and tissue type.

[0012] In some embodiments of the present invention, in step S1, the tissue includes any one of the following: liver, heart, lung, brain, kidney, spleen, intestine, skin, and tumor.

[0013] In some embodiments of the present invention, in step S1, the type of tissue section includes any one of frozen section, paraffin section, and resin section.

[0014] Preferably, the frozen sections include OCT-embedded sections.

[0015] In some embodiments of the present invention, the thickness of the tissue section is 10-30 μm, preferably 20 μm.

[0016] In some embodiments of the present invention, in step S1, the incubation time is 3 to 10 minutes.

[0017] In some embodiments of the present invention, in step S1, the incubation temperature is 0~5°C.

[0018] In some embodiments of the present invention, in step S1, the incubation buffer comprises 8-12 mM Tris-HCl, 8-12 mM NaCl, 3-8 mM MgCl2, 0.01% v / v to 1% v / v micro / nano particles, 0.1-1 U / μL RNase inhibitor, 0.01% w / v to 0.5% w / v bovine serum albumin, and 0.01% v / v to 0.05% v / v nonionic surfactant.

[0019] Preferably, the incubation buffer comprises 8-12 mM Tris-HCl, 8-12 mM NaCl, 3-8 mM MgCl2, 0.01% v / v to 0.5% v / v micro / nano particles, 0.1-0.8 U / μL RNase inhibitor, 0.1% w / v to 0.5% w / v bovine serum albumin, and 0.01% v / v to 0.05% v / v nonionic surfactant.

[0020] In some embodiments of the present invention, in step S1, the micro-nano particles are selected from any one of metal oxide particles, silicon dioxide particles, and colloidal inorganic particles.

[0021] In some embodiments of the present invention, in step S1, the particle size of the micro / nano particles is 10 nm to 100 nm. For example, it can be 10, 20, 30, 50, 100 nm, etc.

[0022] Microparticles can break cell membranes and release cell nuclei by colliding with them, but if the particle size of microparticles is too large, they will generate excessive mechanical force, which may increase the risk of cell nucleus damage.

[0023] In some embodiments of the present invention, in step S1, the micro-nano particles include at least one of metal oxide particles, silicon dioxide particles, and colloidal inorganic particles. Preferably, the metal oxide particles include any one of titanium oxide particles, zinc oxide particles, aluminum oxide particles, iron oxide particles, cerium oxide particles, zirconium oxide particles, copper oxide particles, and magnesium oxide particles.

[0024] Preferably, the colloidal inorganic particles comprise polystyrene (PS) microspheres.

[0025] In some embodiments of the present invention, depending on actual needs, the surface of the micro / nano particles may also be modified with PEG, PVP, BSA, carboxylation, amination, APTES, or polysaccharide coating.

[0026] In this invention, the micro / nanoparticles are preferably hydrophilic and have a density greater than that of the cell nucleus, so as to avoid them interfering with subsequent sequencing.

[0027] In some embodiments of the present invention, in step S1, the nonionic surfactant includes Tween-20, Tween-80, Triton X-100, and Brij. 35. At least one of NP40.

[0028] In some embodiments of the present invention, in step S3, the sieve aperture of the filter is 30~50μm.

[0029] In some embodiments of the present invention, in step S3, the temperature of the centrifugation is 0~5°C; Preferably, the centrifugal force of the centrifugation is 500~1000 g; Preferably, the centrifugation time is 5 to 12 minutes.

[0030] In some embodiments of the present invention, in step S3, the blowing can be performed using a pipette. Blowing with a pipette can apply a gentle and controllable physical disturbance to the system, causing the particles to exert a mechanical effect to promote the release of cell nuclei; the number of blows can be appropriately adjusted according to the fragility of the tissue.

[0031] In some embodiments of the present invention, in step S3, the resuspension includes resuspension with phosphate buffer or water.

[0032] By gently resuspending the precipitate with a suitable nuclear resuspension solution, a single-cell nuclear suspension can be obtained for direct use in downstream experiments.

[0033] In some embodiments of the present invention, the single-cell nucleus dissociation method is preferably performed at 0-5°C or on ice to protect the cell nucleus and RNA from degradation.

[0034] A second aspect of the present invention provides the application of the single-cell nucleus dissociation method as described in the first aspect in the preparation of cell nucleus suspensions.

[0035] A third aspect of the present invention provides a detection device comprising: Processing module for running the single-cell nucleus dissociation method as described in the first aspect; The detection module is used to perform sequencing analysis on the single cell nuclei obtained by the processing module.

[0036] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a microscopic observation image of the single-cell nucleus suspension prepared in Example 1 of the present invention; Figure 2 This is a microscopic observation image of the single-cell nucleus suspension prepared in Example 2 of the present invention; Figure 3 This is a microscopic observation image of the single-cell nucleus suspension prepared in Example 3 of the present invention; Figure 4 This is a microscopic observation image of the single-cell nucleus suspension prepared in Comparative Example 1 of this invention; Figure 5 This is a microscopic observation image of the single-cell nucleus suspension prepared in Comparative Example 2 of this invention. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0039] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0040] When a range of values ​​is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0041] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0042] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] Example 1: Mouse brain tissue slices This embodiment provides a method for single-cell nucleus dissociation based on mouse brain tissue slices, specifically including the following steps: (1) Obtain the sample to be extracted: Mouse brain tissue blocks were placed in a cryoform mold, and then pre-cooled OCT embedding medium was slowly added to completely submerge the tissue, avoiding the formation of air bubbles. The mold was held with pre-cooled forceps and vertically immersed in isopentane with added liquid nitrogen, and kept for 10-30 seconds until the OCT completely turned white and solidified. The frozen block was then removed from -80°C and placed in the sample head of a cryostat (-20°C) for sectioning to obtain mouse brain tissue sections with a thickness of 20 μm.

[0044] (2) Dissociation of the cell nucleus: The 1 mg mouse brain tissue slices obtained above were mounted on a glass slide, and 200 μL of incubation buffer was added to the surface of the slices. The slides were incubated on ice for 5 min. Then, the incubation buffer and the mouse brain tissue slices were transferred together to a 1.5 mL centrifuge tube using a pipette. The tubes were gently pipetted 30 times to obtain the initial nucleus dissociation solution.

[0045] The incubation buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.5% v / v PS microspheres, 0.5 U / μl RNase inhibitor, 0.2% w / v bovine serum albumin, and 0.02% v / v Tween-20. The PS microspheres had a particle size of 50 nm.

[0046] (3) Purification: The initial solution of cell nuclei dissociation was filtered through a 40 μm cell sieve, and the filtrate was collected. The solution was then centrifuged at 4 °C and 1000 g for 10 min, and the precipitate was collected. The precipitate was then resuspended in 50 μL of PBS buffer to obtain a cell nucleus suspension.

[0047] The above-mentioned cell nucleus suspension was stained with trypan blue solution, and the state of the cell nuclei was observed under a microscope. The results are as follows: Figure 1 As shown, a large number of single cell nuclei can be observed, with few impurities and a clean background.

[0048] Furthermore, the nuclei were counted using a hemocytometer, with each group repeated three times. The counting results showed that the number of individual cell nuclei obtained using the nucleus dissociation method of this embodiment was approximately 400 per μL.

[0049] Example 2: Human glioma tissue This embodiment provides a method for single-cell nucleus dissociation based on human glioma tissue sections, specifically including the following steps: (1) Obtain the sample to be extracted: The human glioma tissue block was placed in a cryomold, and then pre-cooled OCT embedding medium was slowly added to completely submerge the tissue, avoiding the formation of air bubbles. The mold was held with pre-cooled forceps and vertically immersed in isopentane with added liquid nitrogen, and kept for 10-30 seconds until the OCT completely turned white and solidified. The frozen block was then removed from -80°C and placed in the sample head of a cryostat (-20°C) for sectioning to obtain human glioma tissue sections with a thickness of 20 μm.

[0050] (2) Dissociation of the cell nucleus: The 1 mg human glioma tissue slices obtained above were mounted on a glass slide, and 200 μL of incubation buffer was added to the surface of the slices. The slides were incubated on ice for 5 min. Then, the incubation buffer and the human glioma tissue slices were transferred together to a 1.5 mL centrifuge tube using a pipette. The tubes were gently pipetted 30 times to obtain the initial nucleus dissociation solution.

[0051] The incubation buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.1% v / v TiO2 nanoparticles, 0.5 U / μl RNase inhibitor, 0.2% w / v bovine serum albumin, and 0.02% v / v Tween-20. The TiO2 nanoparticles had a particle size of 20 nm.

[0052] (3) Purification: The initial solution of cell nuclei dissociation was filtered through a 40 μm cell sieve, and the filtrate was collected. The solution was then centrifuged at 4 °C and 1000 g for 10 min, and the precipitate was collected. The precipitate was then resuspended in 50 μL of PBS buffer to obtain a cell nucleus suspension.

[0053] The above-mentioned cell nucleus suspension was stained with trypan blue solution, and the state of the cell nuclei was observed under a microscope. The results are as follows: Figure 2 As shown, a high content of single cell nuclei can be observed, with few impurities and a clean background, indicating that the method for dissociating cell nuclei from human glioma tissue sections using the present invention can obtain high-quality single cell nuclei.

[0054] Furthermore, the nuclei were counted using a hemocytometer, with each group repeated three times. The counting results showed that the number of individual cell nuclei obtained using the nuclear dissociation method of the present invention was approximately 450 per μL.

[0055] Example 3: Crab-eating monkey brain tissue This embodiment provides a method for single-cell nucleus dissociation based on cynomolgus monkey brain tissue slices, specifically including the following steps: (1) Obtain the sample to be extracted: Place the cynomolgus monkey brain tissue block into a cryomold, then slowly add pre-cooled OCT embedding medium to completely submerge the tissue, avoiding the formation of air bubbles. Hold the mold with pre-cooled tweezers and vertically immerse it in isopentane with added liquid nitrogen, holding for 10-30 seconds until the OCT completely turns white and solidifies. Then remove the frozen block from -80°C and place it in the sample head of a cryostat (-20°C) for sectioning, obtaining cynomolgus monkey brain tissue sections with a thickness of 20 μm.

[0056] (2) Dissociation of the cell nucleus: The 1 mg cynomolgus brain tissue slices obtained above were mounted on a glass slide, and 200 μL of incubation buffer was added to the surface of the slices. The slides were incubated on ice for 5 min. Then, the incubation buffer and the cynomolgus brain tissue slices were transferred together to a 1.5 mL centrifuge tube using a pipette. The tubes were gently pipetted 30 times to obtain the initial nucleus dissociation solution.

[0057] The incubation buffer contains 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.5% v / v polymer-coated colloidal silica nanoparticles, 0.5 U / μl RNase inhibitor, 0.2% w / v bovine serum albumin, and 0.02% v / v Tween-20.

[0058] The polymer-coated colloidal silica nanoparticles were purchased from Sigma-Aldric, catalog number 420786, with a particle size of 22 nm.

[0059] (3) Purification: The initial solution of cell nuclei dissociation was filtered through a 40 μm cell sieve, and the filtrate was collected. The solution was then centrifuged at 4 °C and 1000 g for 10 min, and the precipitate was collected. The precipitate was then resuspended in 50 μL of PBS buffer to obtain a cell nucleus suspension.

[0060] The above-mentioned cell nucleus suspension was stained with trypan blue solution, and the state of the cell nuclei was observed under a microscope. The results are as follows: Figure 3 As shown, a high content of single cell nuclei can be observed, with few impurities and a clean background, indicating that the method for dissociating cell nuclei from cynomolgus monkey brain tissue sections according to the present invention can obtain high-quality single cell nuclei.

[0061] Furthermore, the nuclei were counted using a hemocytometer, with each group repeated three times. The counting results showed that the number of individual cell nuclei obtained using the nuclear dissociation method of the present invention was approximately 500 per μL.

[0062] Comparative Example 1: Mouse brain tissue This comparative example provides a method for single-cell nucleus dissociation based on mouse brain tissue slices, specifically including the following steps: (1) Obtain the sample to be extracted: Mouse brain tissue blocks were placed in a cryoform mold, and then pre-cooled OCT embedding medium was slowly added to completely submerge the tissue, avoiding the formation of air bubbles. The mold was held with pre-cooled forceps and vertically immersed in isopentane with added liquid nitrogen, and kept for 10-30 seconds until the OCT completely turned white and solidified. The frozen block was then removed from -80°C and placed in the sample head of a cryostat (-20°C) for sectioning to obtain mouse brain tissue sections with a thickness of 20 μm.

[0063] (2) Dissociation of the cell nucleus: The 1 mg mouse brain tissue slices obtained above were mounted on a glass slide, and 200 μL of incubation buffer was added to the surface of the slices. The slides were incubated on ice for 5 min. Then, the incubation buffer and the mouse brain tissue slices were transferred together to a 1.5 mL centrifuge tube using a pipette. The tubes were gently pipetted 30 times to obtain the initial nucleus dissociation solution.

[0064] The incubation buffer contains 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, and 5 mM MgCl₂. 2、 0.5 U / μl RNase inhibitor, 0.2% w / v bovine serum albumin and 0.02% v / v Tween-20.

[0065] (3) Purification: The initial solution of cell nuclei dissociation was filtered through a 40 μm cell sieve, and the filtrate was collected. The solution was then centrifuged at 4 °C and 1000 g for 10 min, and the precipitate was collected. The precipitate was then resuspended in 50 μL of PBS buffer to obtain a cell nucleus suspension.

[0066] The above-mentioned cell nucleus suspension was stained with trypan blue solution, and the state of the cell nuclei was observed under a microscope. The results are as follows: Figure 4 As shown, the cells exhibit nuclear aggregation and increased fragmentation, with a significant reduction in the number of individual nuclei.

[0067] Furthermore, the nuclei were counted using a hemocytometer, with each group repeated three times. The counting results showed that the number of cell nuclei obtained using the nucleus dissociation method of the present invention was approximately 190 per μL.

[0068] Comparative Example 2: Mouse brain tissue This comparative example provides a method for single-cell nucleus dissociation based on mouse brain tissue slices, specifically including the following steps: (1) Obtain the sample to be extracted: Mouse brain tissue blocks were placed in a cryomold, and then pre-cooled OCT embedding medium was slowly added to completely submerge the tissue, avoiding the formation of air bubbles. The mold was held with pre-cooled forceps and vertically immersed in isopentane with added liquid nitrogen, and kept for 10-30 seconds until the OCT completely turned white and solidified. The frozen block was then removed from -80°C and placed in the sample head of a cryostat (-20°C) for sectioning to obtain mouse brain tissue sections with a thickness of 20 μm.

[0069] (2) Dissociation of the cell nucleus: The 1 mg mouse brain tissue slices obtained above were mounted on a glass slide, and 200 μL of incubation buffer was added to the surface of the slices. The slides were then incubated on ice for 5 min. The incubation buffer and the mouse brain tissue slices were then transferred together to a 1.5 mL centrifuge tube using a pipette and vortexed for 30 s to obtain the initial nucleus dissociation solution.

[0070] The incubation buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.5 U / μl RNase inhibitor, 0.2% w / v bovine serum albumin, and 0.02% v / v Tween-20.

[0071] (3) Purification: The initial solution of cell nuclei dissociation was filtered through a 40 μm cell sieve, and the filtrate was collected. The solution was then centrifuged at 4 °C and 1000 g for 10 min, and the precipitate was collected. The precipitate was then resuspended in 50 μL of PBS buffer to obtain a cell nucleus suspension.

[0072] The above-mentioned cell nucleus suspension was stained with trypan blue solution, and the state of the cell nuclei was observed under a microscope. The results are as follows: Figure 5 As shown, there is an increase in nuclear fragmentation and a decrease in the number of nuclei.

[0073] Furthermore, the nuclei were counted using a hemocytometer, with each group repeated three times. The counting results showed that the number of cell nuclei obtained using the nuclear dissociation method of the present invention was approximately 60 per μL.

[0074] In summary, this invention provides a method and application for single-cell nucleus dissociation suitable for tissue sections. This invention achieves gentle shearing of the cell membrane through micro / nano-particle assistance and controllable physical perturbation, effectively releasing the cell nucleus while maintaining nuclear membrane integrity and reducing the risk of nuclear damage and RNA degradation. Furthermore, the cell nucleus dissociation method of this invention is simple and the operational intensity is easily and precisely controlled, significantly reducing the risk of nuclear rupture and fragmentation. It enables efficient and low-damage preparation of single-cell nucleus suspensions, offering significant advantages over traditional chemical lysis or mechanical shearing methods in terms of nuclear integrity, RNA quality, and sample compatibility.

[0075] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for dissociating single-cell nuclei, characterized in that, Includes the following steps: S1. Obtain the tissue sections to be processed, mount them on the carrier surface, and then add incubation buffer for incubation; The incubation buffer contains Tris-HCl, NaCl, MgCl2, micro / nano particles, RNase inhibitors, bovine serum albumin, and nonionic surfactants. S2. Transfer the incubated product to a centrifuge tube and gently blow it 5-45 times to obtain the initial solution of cell nucleus dissociation. S3. Filter and centrifuge the initial solution of cell nucleus dissociation, collect the precipitate, and resuspend it to obtain a cell nucleus suspension.

2. The method for single-cell nucleus dissociation according to claim 1, characterized in that, In step S1, the tissue includes any one of the following: liver, heart, lung, brain, kidney, spleen, intestine, skin, and tumor; And / or, the type of tissue section includes any one of frozen section, paraffin section, and resin section; And / or, the thickness of the tissue section is 10~30μm.

3. The method for single-cell nucleus dissociation according to claim 2, characterized in that, In step S1, the incubation time is 3 to 10 minutes; And / or, the incubation temperature is 0~5℃.

4. The method for single-cell nucleus dissociation according to claim 1, characterized in that, In step S1, the incubation buffer contains 8-12 mM Tris-HCl, 8-12 mM NaCl, 3-8 mM MgCl2, 0.01% v / v ~ 1% v / v micro / nano particles, 0.1-1 U / μL RNase inhibitor, 0.01% w / v ~ 0.5% w / v bovine serum albumin, and 0.01% v / v ~ 0.05% v / v nonionic surfactant.

5. The method for single-cell nucleus dissociation according to claim 1, characterized in that, In step S1, the micro-nano particles are selected from any one of metal oxide particles, silica particles, and colloidal inorganic particles; And / or, the particle size of the micro / nano particles is 10 nm to 100 nm; And / or, the metal oxide particles include any one of titanium oxide particles, zinc oxide particles, aluminum oxide particles, iron oxide particles, cerium oxide particles, zirconium oxide particles, copper oxide particles, and magnesium oxide particles; And / or, the colloidal inorganic particles include polystyrene microspheres.

6. The method for single-cell nucleus dissociation according to claim 1, characterized in that, In step S1, the nonionic surfactants include Tween-20, Tween-80, Triton X-100, and Brij.

35. At least one of NP40.

7. The method for single-cell nucleus dissociation according to any one of claims 1 to 6, characterized in that, In step S3, the sieve aperture of the filter is 30~50μm.

8. The method for single-cell nucleus dissociation according to claim 7, characterized in that, In step S3, the centrifugation temperature is 0~5℃; And / or, the centrifugal force of the centrifugation is 500~1000 g; And / or, the centrifugation time is 5~12 min.

9. The application of the single-cell nucleus dissociation method according to any one of claims 1 to 8 in the preparation of cell nucleus suspensions.

10. A detection device, characterized in that, include: The processing module is used to run the single-cell nucleus dissociation method as described in any one of claims 1 to 8; The detection module is used to perform sequencing analysis on the single cell nuclei obtained by the processing module.

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