A method for single nuclei isolation of hair-rich skin samples

Abstract

The present application provides a method for isolating single nuclei of a hair-rich skin sample. Specifically, the isolation method of the present application uses a combination of lysate components capable of digesting dissociated hair-rich skin tissue, optimizes the lysing time of the lysate, the frequency and duration of tissue grinding, and uses magnetic bead adsorption of nuclei to remove debris impurities. The method of the present application can cover all cell types in the tissue sample without bias, remove impurities sufficiently, and has the prospect of application in the field of single cell research.

Description

Technical Field

[0001] This invention relates to the field of cell biology. Specifically, this invention relates to a method for single-cell nucleus isolation from hair-rich skin samples. Background Technology

[0002] In recent years, the rapid development of single-cell sequencing technology has provided strong support for high-resolution research on tissue heterogeneity and gene expression regulatory networks. However, single-cell sequencing technology has significant limitations. For example, single-cell transcription sequencing cannot be used for testing frozen tissue samples; enzymes' preference for cell type digestion may lead to cell loss; excessively large cells cannot be captured by high-throughput sequencing microfluidic chips or microporous chips; and lipid-rich cells are difficult to capture by existing capture technologies, resulting in cell loss. Single-cell nuclear sequencing technology solves these problems in single-cell sequencing. Single-cell nuclear technology mainly lyses the cell membrane by combining mechanical grinding and enzymatic digestion of the tissue. Because the nuclear membrane is relatively robust, under certain conditions, the cell nucleus remains intact after the cell membrane ruptures. The cell nucleus contains the genome and RNA with a high degree of similarity to that in the cytoplasm. At the same time, the biological properties of the nucleus are relatively homogeneous across different cell types, so it can be used to replace the genetic information in the cytoplasm. Dissociation of cell nuclei is unaffected by whether the sample is cryopreserved, enzymes' preference for cell type digestion, cell size, or lipid cells. After dissociation of cell nuclei, relevant information of all cell types can be captured and recovered for unbiased analysis, providing a complete reflection of cellular and molecular regulatory information in tissues. Therefore, it is an important supplement to single-cell technology.

[0003] Single-cell technology has been applied in hair follicle research. Current research on hair follicles is still incomplete, especially regarding the mechanisms by which signal interactions between different cells in the hair follicle affect the hair cycle and hair growth. Single-cell technology provides a powerful tool for high-resolution studies of cellular heterogeneity and gene expression differences at the single-cell level. Several reports have documented the use of single-cell technology to analyze hair follicles. However, due to the inherent limitations of current high-throughput single-cell techniques, when tissue samples are dissociated into single-cell suspensions, lipid cells cannot be captured by current library preparation methods. This results in the loss of lipid cells in the hair follicle, including adipocytes and other lipid-rich cell types, due to methodological limitations, making it impossible to analyze their role in the hair follicle. Another approach to capture all cell types is single-nucleus technology, which uses enzymatic digestion, grinding, and other methods to separate the cell nuclei from the cells. The nuclei of lipid cells can also be separated and captured, thus avoiding the natural loss of lipid cells in conventional single-cell techniques. However, for samples rich in hair follicles, hair roots are deeply embedded in the follicle tissue and cannot be pre-removed. Therefore, during single-cell nucleus separation, hair debris inevitably gets mixed in with the final suspension and cannot be effectively separated from the nuclei. This results in low single-cell nucleus capture efficiency, excessive impurity content, and substandard library construction. Therefore, developing single-cell related technologies for hair-rich samples is of great significance for studying and understanding the composition of hair follicles and their interactions.

[0004] Currently, single-cell and single-nucleus techniques for general tissues are unsuitable for processing tissue samples rich in impurities such as hair due to technical limitations. The main problems include: 1) Single-cell transcriptomics naturally loses lipid-rich cells during tissue sample dissociation, making it impossible to study the role of adipocytes and other cells in the hair follicle microenvironment; 2) While single-nucleus techniques can capture nuclei of all cell types, hair and other components inevitably generate debris during the separation process, leading to contamination and preventing the effective separation of pure nuclei for subsequent analysis. These limitations of existing technologies prevent the current ability to perform cell and molecular interaction analyses covering all cell types in samples rich in hair and other impurities.

[0005] Therefore, there is a need in the art to develop a method for isolating single cell nuclei from tissue samples rich in impurities such as hair, thereby reducing interference from impurities on the isolated cell nuclei. Summary of the Invention

[0006] The purpose of this invention is to provide a method for single-cell nucleus separation in hair-rich skin samples.

[0007] In a first aspect of the present invention, a method for isolating single cell nuclei from a tissue is provided, the method comprising the steps of:

[0008] (1) A tissue sample is provided, and the tissue sample is lysed in a lysis buffer to obtain a first suspension, wherein the lysis buffer contains or is composed of components of group (A) or group (B):

[0009] (A) Trypsin and NP-40;

[0010] (B) Collagenase I, collagenase IV, dispase, and trypsin;

[0011] (2) The first suspension is digested using the lysis buffer to obtain the second suspension;

[0012] (3) Filter the second suspension to remove tissue blocks, thereby obtaining the third suspension;

[0013] (4) Add magnetic beads that specifically bind to cell nuclei to the third suspension, separate the magnetic beads, and thus obtain the separated cell nuclei.

[0014] In another preferred embodiment, the tissue sample is a skin sample containing hair follicles.

[0015] In another preferred embodiment, in step (1), the tissue sample is a fresh sample or a frozen sample.

[0016] In another preferred embodiment, step (1) includes the step:

[0017] (1a) The tissue sample was minced in the lysis buffer;

[0018] (1b) Grind the tissue sample using a grinding pestle and then grind it using a grinder to obtain the first suspension.

[0019] In another preferred embodiment, in step (1b), the tissue sample is ground using a grinding pestle for 3-5 minutes.

[0020] In another preferred embodiment, in step (1b), the grinding frequency is 20-80Hz (preferably 30-60Hz, more preferably 45-50Hz).

[0021] In another preferred embodiment, in step (1b), the grinding time of the grinder is 30s-5min, more preferably 1min-3min, and even more preferably 2min-2.5min.

[0022] In another preferred embodiment, the lysis buffer comprises, or is composed of, the following components:

[0023] (A) 0.05%-1% trypsin (preferably 0.25%-0.5%) and

[0024] 0.001%-0.1% NP-40 (preferably 0.01%-0.03%)

[0025] Measured as a percentage of mass-volume concentration.

[0026] In another preferred embodiment, the lysis buffer comprises, or is composed of, the following components:

[0027] (B) 0.5-10 mg / kg collagenase I (preferably 2-4 mg / kg),

[0028] 0.5-10 mg / kg collagenase IV (preferably 2-4 mg / kg),

[0029] 0.5-10 mg / kg dispersant enzyme (preferably 2-4 mg / kg) and

[0030] 0.025%-0.5% trypsin (preferably 0.125%-0.25%)

[0031] Measured as a percentage of mass-volume concentration.

[0032] In another preferred embodiment, the solvent of the lysis buffer is a phosphate buffered saline (PBS).

[0033] In another preferred embodiment, in step (2), the first suspension is digested at 0-10°C (preferably 4-5°C) for 10-60 min (preferably 30-40 min).

[0034] In another preferred embodiment, in step (2), the container is inverted every 5 minutes during digestion.

[0035] In another preferred embodiment, in step (3), a second suspension is filtered using a 40µm cell filter to remove tissue blocks.

[0036] In another preferred embodiment, step (4) includes the step:

[0037] (4a) Centrifuge the third suspension, remove the supernatant, and retain the precipitate;

[0038] (4b) Resuspend the precipitate using a cell nucleus preservation solution;

[0039] (4c) Add nucleus-binding magnetic beads to the resuspension and let stand to allow the magnetic beads to bind to the cell nucleus;

[0040] (4d) Adsorb magnetic beads until the resuspension is clear, remove the supernatant, and retain the adsorbed magnetic beads;

[0041] (4e) Wash and resuspend the magnetic beads, adsorb the magnetic beads again, and remove the supernatant;

[0042] (4f) Add the reaction termination elution solution and let stand to separate the cell nucleus from the magnetic beads. After the magnetic beads are adsorbed, the supernatant after adsorption is aspirated to obtain the separated cell nucleus.

[0043] In another preferred embodiment, the nuclear-binding magnetic beads are concanavalin A-binding magnetic beads, more preferably BioMagPlus Concanavalin A beads.

[0044] In a second aspect of the invention, an isolated cell nucleus is provided, said cell nucleus being isolated by means of the method described in the first aspect of the invention.

[0045] In another preferred embodiment, the cell nucleus is isolated from a skin sample containing hair follicles.

[0046] In another preferred embodiment, the cell nucleus comprises a cell nucleus derived from an adipocyte.

[0047] In a third aspect of the invention, the application of isolated cell nuclei as described in the second aspect of the invention is provided in the construction of intranuclear genomic libraries, epigenome libraries, intranuclear transcriptome libraries, protein detection, metabolite detection, cell nucleus transplantation, or cell nucleus-related function detection.

[0048] In a fourth aspect of the invention, a lysis buffer is provided for lysing hair-containing tissue, said lysis buffer comprising, or consisting of, components of group (A) or group (B):

[0049] (A) Trypsin and NP-40;

[0050] (B) Collagenase I, collagenase IV, dispase, and trypsin.

[0051] In another preferred embodiment, the solvent of the lysis buffer is a phosphate buffered saline (PBS).

[0052] In another preferred embodiment, the lysis buffer comprises, or is composed of, the following components:

[0053] (A) 0.05%-1% trypsin (preferably 0.25%-0.5%) and

[0054] 0.001%-0.1% NP-40 (preferably 0.01%-0.03%)

[0055] Measured as a percentage of mass-volume concentration.

[0056] In another preferred embodiment, the lysis buffer comprises, or is composed of, the following components:

[0057] (B) 0.5-10 mg / kg collagenase I (preferably 2-4 mg / kg),

[0058] 0.5-10 mg / kg collagenase IV (preferably 2-4 mg / kg),

[0059] 0.5-10 mg / kg dispersant enzyme (preferably 2-4 mg / kg) and

[0060] 0.025%-0.5% trypsin (preferably 0.125%-0.25%)

[0061] Measured as a percentage of mass-volume concentration.

[0062] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0063] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0064] Figure 1 The effects of tissue digestion at different lysis times are shown.

[0065] Figure 2 The grinding effect of the samples at different grinding machine frequencies is shown.

[0066] Figure 3 The effects of lysis buffers A and B on skin tissue containing hair follicles were shown.

[0067] Figure 4 This demonstrates the effectiveness of the present invention in removing impurities from tissue samples.

[0068] Figure 5 The results of microscopic observation of cell nucleus suspensions obtained after processing skin tissue containing hair follicles using the present invention and prior art are shown.

[0069] Figure 6 The results of qPCR identification of adipocyte marker gene expression in extracted cell nuclear samples are shown. Detailed Implementation

[0070] Through extensive and in-depth research, the inventors have developed, for the first time, a method for single-cell nucleus isolation from hair-rich skin samples. This method effectively removes impurities such as hair generated during the nucleus isolation process, preventing isolation failure. Furthermore, the isolated nuclei retain biological activity and can be used for subsequent multi-omics library construction of single-cell nuclei, including transcriptomics, methylation, chromatin accessibility, and nuclear transplantation. Based on this, the present invention was completed.

[0071] hair follicles

[0072] Mammalian skin consists of several distinct layers, with the epidermis forming the outermost layer and providing the skin barrier function. It lies atop a dermis rich in fibroblasts and collagen. The latter is further subdivided into a thin superior papillary dermis and a thicker inferior reticular dermis, beneath which lies the subcutaneous fat layer, partially integrated into the dermis. In hairy mammals, the skin is traversed by ectodermal appendages: hair follicles and sweat glands. Hair follicles are tiny organs rich in stem cells that repeatedly regenerate new hair during the hair growth cycle. This cycle consists of three phases: active hair growth (anagen phase), regression (catagen phase), and resting (telogen phase). Hair growth is maintained by a dynamic balance between hairs entering the catagen phase and newly entering the anagen phase within the hair follicle, thus ensuring a normal hair quantity.

[0073] Hair follicles are primarily composed of five cell types: keratinocytes, T lymphocytes, myeloid cells, fibroblasts, endothelial cells, and adipocytes. These different cell types cooperate throughout the hair follicle cycle to maintain its dynamic changes. The lower part of the hair follicle, closest to the dermal papilla that induces hair growth, is generally referred to as the proximal end, while the upper part is called the distal end. The hair follicle reaches its maximum size during the anagen phase, at which point its proximal hair bulb penetrates deep into the subcutaneous fat. The hair bulb contains actively dividing epithelial matrix progenitor cells and specialized dermal papilla fibroblasts, which are key signaling centers within the hair follicle. Hair growth is maintained by proliferation and differentiation activities occurring in the hair matrix, while bulging stem cells and progenitor cells are present at the distal end. The hair follicle contains a sebaceous gland that produces sebum. One of the most notable characteristics of the fat in the hair follicle is its ability to periodically remodel in coordination with the hair cycle, including significant thickening of the subcutaneous fat around the hair follicle during the anagen phase, followed by thinning as the hair follicle transitions through the catagen to the telogen phase.

[0074] Single-cell nucleus isolation method

[0075] This invention provides a method for the isolation of single-cell nuclei from tissues rich in impurities such as hair follicles. Specifically, this invention involves mechanically grinding and enzymatically digesting fresh or frozen skin tissue samples containing hair to release cell nuclei, thereby preparing a cell nucleus suspension covering all cell types, including lipid cells. During the mechanical grinding process, hair debris mixed with cell nuclei is added, and magnetic beads specifically designed to adsorb cell nuclei are added. After adsorption using a magnetic frame, the cell nuclei are encapsulated and fixed to the tube wall, while impurities remain in the suspension. The suspension is then removed, a resuspension is added, and the magnetic frame is removed, thereby releasing the cell nuclei back into the suspension, achieving the purpose of purifying the cell nuclei and removing impurities.

[0076] The technical solution of this invention can, on the one hand, cover all cell types in tissues rich in impurities such as hair without bias, and on the other hand, effectively purify cell nuclear suspensions, remove impurities, and obtain cell nuclear suspension samples that can be used for subsequent high-throughput sequencing at the single-cell nucleus level. This provides a technical solution for comprehensive and in-depth analysis of the cell and molecular interaction and regulatory networks of such samples, as well as for elucidating the pathogenesis of related diseases and identifying intervention targets.

[0077] The main advantages of this invention include:

[0078] 1) This invention provides a composition of lysis buffer components for digesting and dissociating hair-rich skin tissue. Testing has shown that the lysis buffer of this invention effectively dissociates cells, thoroughly digesting cells at the hair root, ensuring that all cell types are covered without loss of any specific cell type.

[0079] 2) This invention optimizes the lysis time of the lysis buffer, so that the cells are not over-digested and the tissue is not incompletely dissociated, resulting in the loss of certain cell types.

[0080] 3) This invention optimizes the frequency and duration of tissue grinding for dissociating hair-rich skin tissue. Tests showed that grinding at 45Hz for 2 minutes yielded the best results.

[0081] 4) This invention employs a method that uses magnetic beads to adsorb cell nuclei, thereby removing debris and impurities. It is suitable for purifying cell nuclei from special tissues containing impurities, such as those rich in hair.

[0082] 5) The method of the present invention can cover all cell types in tissue samples without bias. The presence of lipid cells in the isolated cells was confirmed by qPCR detection of lipid cell markers, and a purified single-cell suspension that can meet the requirements of various subsequent tests was obtained.

[0083] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are mass-volume percentages (1% means 1 g of substance in 100 ml of solution) and weight parts.

[0084] Existing technology

[0085] Currently, the main method for isolating single cell nuclei from tissue samples for high-throughput sequencing is the nucleus isolation protocol from 10X Genomics. Its main workflow includes:

[0086] 1) Tissue cryopreservation. Tissues should be rinsed with 1x PBS to remove blood and other debris, patted dry as soon as possible after harvesting, and then frozen. For rapid freezing (also known as quick freezing), place the tissues in test tubes for long-term storage (cryotubes or Eppendorf tubes with a paraffin-coated top), then immerse the tubes in liquid nitrogen or a liquid nitrogen cooling bath (such as isopentane), or deeply place the tubes in a dry ice bucket. Wait at least 2-3 minutes for the tissues to freeze completely, then transfer the tubes containing the tissues to gaseous liquid nitrogen for long-term storage. For short-term storage, tissues can be stored at -80°C.

[0087] 2) Dissociation of cryopreserved tissue. Weigh the tissue before dissociation, add 200 μl of lysine buffer, take 3-50 mg of tissue, hold the pestle and dissociate the tissue in the lysine buffer. Twist the pestle continuously in the test tube, alternately sliding the tissue up and down between the tube wall and the pestle until no large pieces of tissue remain in the lysate. Crush the cryopreserved tissue to form a homogeneous mixture.

[0088] 3) Isolate the cell nucleus. Add 300 μl of lysine buffer and incubate on ice for 10 minutes. Transfer the isolated tissue to the assembled nucleic acid separation column and collection tube using a pipette, centrifuge at 16000 rcf at 4°C for 20 seconds, and pass the homogenized tissue through the provided centrifuge column to separate the cell nucleus from the broken sample.

[0089] 4) Removal of cell debris. Purify the nuclear suspension using the provided cell debris removal buffer and wash buffer. Discard the separation column, vortex for 10 seconds to resuspend, centrifuge at 500 rcf at 4°C for 3 minutes, discard the supernatant, and resuspend the particles in 500 μl of debris removal buffer. Centrifuge at 700 rcf at 4°C for 10 minutes, and discard the supernatant. Resuspend the cell nuclei in 1 ml of wash buffer. Centrifuge at 500 rcf at 4°C for 5 minutes, discard the supernatant, and repeat the elution with debris removal buffer.

[0090] 5) Resuspend cell nuclei. Add 50-500 μl of resuspending buffer, vortex for 3 seconds to resuspend cell nuclei, and check the quality of cell nuclei for instrumental analysis.

[0091] Example 1: Optimization of hair follicle cell nucleus separation process

[0092] 1. Pyrolysis time

[0093] To obtain cells from tissues, tissue samples need to be ground and lysed. Previous studies have found that enzymatic hydrolysis temperature, enzyme concentration, and incubation time are the most critical factors in preparing single-cell suspensions. Changes in these factors can lead to fluctuations in cell viability, cell yield, cell integrity, and gene expression integrity.

[0094] The conditions for lysing tissue samples by grinding in lysis buffer followed by digestion for 15 min, 20 min, 25 min, 30 min, 1 h, and 2 h were tested, and the results are as follows: Figure 1 As shown, after grinding and digestion for about 15 minutes, the tissue still appears as clumps, with hair remaining within the tissue, indicating incomplete digestion and potential loss of certain cell types, especially those close to the hair root. Digestion for more than 30 minutes after grinding yields completely digested hair that is fully separated from the tissue, indicating thorough digestion. To preserve the activity of nucleic acids in the cell nucleus as much as possible, a minimum digestion time of 30 minutes was used.

[0095] 2. Grinding machine frequency

[0096] The grinding effect was tested at 25Hz, 45Hz, and 65Hz for 2 minutes. For example... Figure 2 As shown, grinding at 25Hz still resulted in numerous clumps, indicating poor dissociation. Grinding at 65Hz produced a large amount of debris, possibly due to over-dispersion. Grinding at 45Hz yielded a well-separated sample with less debris.

[0097] Example 2: Isolation of hair follicle cell nuclei

[0098] Experimental steps:

[0099] 1) Skin samples containing intact hair follicles were surgically extracted, stored in DMEM culture medium, and transported.

[0100] 2) For fresh samples, wash the tissue once with PBS after removing it from the culture medium;

[0101] 3) Place the tissue in an EP tube containing 1 ml of lysis buffer and cut it into small pieces with scissors; for frozen samples, place them directly into an EP tube containing 1 ml of lysis buffer and cut them into small pieces.

[0102] 4) Grind with a grinding pestle for 3-5 minutes, then put the sample suspension into a tissue grinder and grind at 45 Hz for 2 minutes.

[0103] 5) Place the ground sample on ice for 30 minutes to digest. Invert the EP tube containing the tissue every 5 minutes to mix.

[0104] 6) Filter the lysate containing tissue using a 40µm cell filter to remove impurities and tissue fragments;

[0105] 7) Centrifuge at 2000g for 5 minutes at 4℃, and carefully remove the supernatant;

[0106] 8) Resuspend the precipitate in 200 μL of nuclear preservation solution. A large amount of black hair debris impurities can be seen in the precipitate. Figure 4 );

[0107] 9) Add 20 μL of BioMag Plus Concanavalin A beads, mix well, and let stand at room temperature for 20 minutes;

[0108] 10) Place the EP tube on the magnetic rack until the liquid becomes clear, then remove the liquid;

[0109] 11) Clean the magnetic beads once with wash buffer, use a magnetic holder to attract the tube, and remove the supernatant;

[0110] 12) Resuspend the beads in 100 μL wash buffer, then add 100 μL reaction termination eluent, mix well, and let stand for 20 min.

[0111] 13) Adsorb the beads with a magnetic rack, collect the supernatant, and the cell nuclei are in the supernatant.

[0112] The lysis buffer components in this method are: Solution A: 0.25% trypsin + 0.01% NP-40; Solution B: 2 mg / ml collagenase I, IV, Dispase, and 0.125% trypsin. Both lysis buffers were tested and found to be effective.

[0113] Experimental results:

[0114] 1. The cleavage effect of different component lyases

[0115] The lysis effect of using solution A (0.25% trypsin + 0.01% NP-40) is as follows: Figure 3 As shown in Figure A, the tissue at the hair root has been effectively dissociated, preventing cell type loss due to incomplete lysis.

[0116] The pyrolysis effect of using solution B is as follows: Figure 3 As shown in B, it can also effectively and completely lyse hair tissue. In comparison, the product obtained from lysis buffer A is purer.

[0117] 2. Impurity removal effect

[0118] like Figure 4 As shown, the above technical solution can effectively purify cell nucleus suspensions and remove the influence of impurities.

[0119] 3. Comparison of the technical solution of this invention with the prior art

[0120] In contrast to existing single-cell isolation methods (10X Genomics), lipid cells cannot be captured due to their low density, resulting in the loss of native cell types. Furthermore, in existing single-nucleus isolation methods, the isolated nuclei cannot be cleaned of hair debris and other impurities, thus failing to meet the library construction quality requirements for subsequent detection. Figure 5 A).

[0121] This method isolates and purifies hair-rich tissue cell nuclei, effectively removing impurities from the final cell nucleus suspension. Figure 5 B). Since RNA expression in the cell nucleus is highly similar to that in the cytoplasm, qPCR was used to identify the expression of adipocyte marker genes in the obtained nuclear samples. The results showed the expression of adipocyte marker genes FABP4 and PPARg in the samples. Figure 6 This confirms that the separation method of the present invention preserves adipocytes, and the final purified cell nucleus sample contains cell nuclei derived from adipocytes.

[0122] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for isolating single cell nuclei from tissue, characterized in that, The method includes the following steps: (1) A tissue sample is provided, and the tissue sample is lysed in a lysis buffer to obtain a first suspension, wherein the lysis buffer contains or is composed of components of group (A) or group (B): (A) Trypsin and NP-40; (B) Collagenase I, collagenase IV, dispase, and trypsin; (2) The first suspension is digested using the lysis buffer to obtain the second suspension; (3) Filter the second suspension to remove tissue blocks, thereby obtaining the third suspension; (4) Add magnetic beads that specifically bind to cell nuclei to the third suspension, separate the magnetic beads, and thus obtain the separated cell nuclei.

2. The method as described in claim 1, characterized in that, The tissue sample mentioned is a skin sample containing hair follicles.

3. The method as described in claim 1, characterized in that, Step (1) includes the following steps: (1a) The tissue sample was minced in the lysis buffer; (1b) The tissue sample is ground using a grinding pestle and then ground using a grinder to obtain the first suspension, wherein the grinder frequency is 20-80 Hz (preferably 30-60 Hz).

4. The method as described in claim 3, characterized in that, In step (1b), the grinding machine frequency is 45-50Hz.

5. The method as described in claim 1, characterized in that, The lysis solution contains, or is composed of, the following components: (A) 0.05%-1% trypsin (preferably 0.25%-0.5%) and 0.001%-0.1% NP-40 (preferably 0.01%-0.03%) Measured as a percentage of mass-volume concentration.

6. The method as described in claim 1, characterized in that, The lysis solution contains, or is composed of, the following components: (B) 0.5-10 mg / ml collagenase I (preferably 2-4 mg / ml), 0.5-10 mg / ml collagenase IV (preferably 2-4 mg / ml), 0.5-10 mg / ml dispersant enzyme (preferably 2-4 mg / ml) and 0.025%-0.5% trypsin (preferably 0.125%-0.25%) Measured as a percentage of mass-volume concentration.

7. The method as described in claim 1, characterized in that, In step (2), the first suspension is digested at 0-10℃ (preferably 4-5℃) for 10-60 min (preferably 30-40 min).

8. An isolated cell nucleus, characterized in that, The cell nucleus is obtained by means of the method described in claim 1.

9. The application of the isolated cell nucleus as described in claim 8 in the construction of intranuclear genomic libraries, epigenome libraries, intranuclear transcriptome libraries, protein detection, metabolite detection, cell nucleus transplantation, or cell nucleus-related function detection.

10. A lysis buffer for lysing hair-containing tissue, characterized in that, The lysis buffer contains, or is composed of, components of group (A) or group (B): (A) Trypsin and NP-40; (B) Collagenase I, collagenase IV, dispase, and trypsin.

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