A method for simultaneous exosome, single-cell, and single-cell nucleus sequencing in the same intrahepatic bile duct tissue sample

Abstract

The present invention discloses a method for preparing exosomes, single cell suspensions and single cell nuclear suspensions based on the same intrahepatic bile duct tissue sample at the same time. The method described in the present invention can obtain more meaningful data in the same sample, dig out deeper biological information, and thus analyze the most critical dynamic mechanism of the network behind the biological phenomenon. The present invention innovatively and improvedly combines the three technologies of exosome preparation, single cell suspension preparation and single cell nuclear suspension preparation, breaking through the existing technical barriers, obtaining a more comprehensive cell type in the sample, and truly analyzing the network mechanism of cell interaction behind life phenomena (not only understanding the function of a single cell, but also understanding how signals are transmitted between cells and how specific cells respond to signals.) This enriches the research data and provides a basis for digging deeper into biological mechanisms.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a method for simultaneously preparing exosomes, single-cell suspensions, and single-cell nucleus suspensions from the same human intrahepatic bile duct tissue sample, a method for simultaneously performing exosome small RNA sequencing, single-cell sequencing, and single-cell nucleus sequencing, as well as a kit for preparing single-cell and single-cell nucleus suspensions and applications thereof. Background Art

[0002] The development of tissues and organs, as well as the body's homeostasis, all rely on communication between different cells. It is through diverse signaling molecules that a wide variety of cells can coordinate with one another in an orderly manner, thus manifesting a diverse range of life phenomena. Therefore, the essence of life is a network of interactions between different cell types. To understand this network, we need to understand not only the functions of individual cells but also how cells transmit signals to each other—the communication signals within the cellular interaction network. Combining these two components allows us to "reconstruct" the complete cellular interaction network, thereby revealing a deeper understanding of the molecular mechanisms underlying life phenomena. Therefore, only by exploring both components simultaneously can we truly uncover the mechanisms underlying this network.

[0003] Since its introduction, single-cell sequencing has garnered widespread attention in both basic research and industry, becoming a highly promising technology. Compared to traditional whole-genome sequencing at the multi-cell level, single-cell sequencing not only measures gene expression more precisely but can also detect trace amounts of gene expression proteins or rare non-coding RNAs, offering comprehensive and multi-faceted advantages. Furthermore, exosomes, nanoscale vesicles secreted by living cells that carry RNA, proteins, lipids, and other components, serve as vectors for intercellular communication and are a natural tool for studying tissue microenvironments and the communication between different cell subpopulations. They are involved in immune responses, cell migration, cell differentiation, and tumor invasion. Single-cell transcriptomes can clarify cellular functions, while exosomal miRNAs can reveal information transmitted between cells. Therefore, simultaneous analysis of exosomal small RNA and single-cell transcriptomes in the same tissue can truly unravel the network mechanisms underlying these cellular interactions.

[0004] For scRNA-seq sequencing, a high-quality single-cell suspension is the lower limit for single-cell sequencing results and the only factor associated with high-quality data. However, due to the 37°C enzymatic digestion process involved in preparing single-cell suspensions, scRNA-seq often results in the loss of cell types that are intolerant to enzymatic digestion, such as epithelial cells, which are often difficult to capture. Fibroblasts and endothelial cells are more embedded in the extracellular matrix and basement membrane, making them even more difficult to digest. Immune cell types, however, are naturally isolated from the tissue environment and are easily accessible through enzymatic digestion. This often results in scRNA-seq data showing a bias toward immune cell types. Furthermore, some sensitive cells may be fragmented due to excessive dissociation. In contrast, snRNA-seq, which uses mechanical and chemical cell disruption, avoids the biases associated with enzymatic digestion methods. All cell types can be effectively recovered and identified, enabling researchers to obtain a more complete and comprehensive cellular landscape that reflects the true proportion of cell types in a tissue, making it a popular method for researchers. However, because immune cells themselves account for a small proportion, and cell-based systems such as 10xGenomics have limitations on cell quantity, snRNA-seq usually captures fewer immune cell types, which has caused great trouble for many researchers who are concerned about immune mechanisms. Although there are reports that the same tissue can be divided into two parts for scRNA-seq and snRNA-seq respectively, this processing method will also produce bias for many samples with great heterogeneity and cannot truly reflect the cellular composition of the tissue, resulting in a significant reduction in the authenticity and accuracy of the results. If a method can be established that can simultaneously perform scRNA-seq and snRNA-seq in the same tissue, after using scRNA-seq to collect more immune cells, and then use snRNA-seq to supplement other cell types, thereby achieving the goal of "more comprehensively capturing cell types in the tissue while also highlighting immune cell types", it can meet the various needs of researchers.

[0005] In summary, there is a need to develop a method that can simultaneously sequence exosomes, single cells, and single nuclei within the same tissue. While scRNA-seq and snRNA-seq can capture a more comprehensive range of cell types to address individual nodes in the network, exosome small RNA sequencing can then be used to decipher the communication signals between nodes, thereby uncovering deeper biological information.

[0006] Intrahepatic cholangiocarcinoma is a type of primary liver cancer, with an incidence rate second only to hepatocellular carcinoma. The main treatment method is surgery, with a poor prognosis, difficulty in curing, and short survival time after surgery. However, in the current existing technology, there is no technical solution that can realize the simultaneous sequencing of exosomes, single cells, and single cell nuclei in the same intrahepatic cholangiocarcinoma. There is a key technical problem that needs to be solved, that is, how to simultaneously separate exosomes, single cell suspensions, and single cell nuclei suspensions from the same tissue. If exo+snRNA-seq&scRNA-seq can be performed in human intrahepatic bile duct tissue at an earlier time point, the signal regulatory network and cell type and corresponding functions in the corresponding TME environment can be found, which will fill the existing gaps, lay the foundation for high-level research, and provide a precise basis for the development of more effective early treatments and even prevention. Summary of the Invention

[0007] In order to overcome the problems existing in the prior art, the present invention innovatively proposes for the first time a method and application for preparing exosomes, single cell suspensions and single cell nuclear suspensions based on the same tissue sample (for example, the same intrahepatic bile duct cancer tissue). Using intrahepatic bile duct tissue for exo+snRNA-seq&scRNA-seq can prepare a single cell suspension with a total cell volume of 98W, a fragmentation rate of 4%, and a clumping rate of 4%, and a single cell nuclear suspension with a total cell nucleus volume of 220W, a fragmentation rate of 5%, and a clumping rate of 5%. The morphology and concentration of exosomes can meet the requirements of small RNA library construction. To date, technical research in this area in the prior art is still in a blank stage and no reports have been seen. The method of the present invention can not only comprehensively obtain the cell types in the intrahepatic bile duct tissue sample, but also conduct a deeper exploration of the communication between the nodes of the cells in the intrahepatic bile duct sample, such as how the cells transmit signals and how specific cells respond to the signals. However, existing technical methods can only detect exosomal miRNA and single-cell (single-cell nucleus) transcriptomes separately, and it is impossible to obtain both types of data simultaneously in the same sample. At present, some studies first find specific cell types through single-cell (nuclear) transcriptomes, and then detect exosomes in the supernatant of these cultured cells through sorting and in vitro culture. However, these exosomes cannot truly reflect the state of cells in tissues, and therefore may cause false positives and omissions. There are also studies that simultaneously identify single-cell exosomes and single-cell (nuclear) transcriptomes, but the same in vitro culture environment makes it difficult to truly reflect the true state of cells in tissues and organs. This greatly limits the organic relationship between the two, making it difficult for them to complement and support each other, resulting in the inability to fully realize the true power of this joint research. At the same time, single-cell sequencing has a preference for dissociating immune cells, and single-cell nucleus sequencing captures fewer immune cells. Therefore, the present invention has created an exo+scRNA-seq&snRNA-seq process, overcoming the technical barriers and difficulties of current technologies that prevent the simultaneous performance of small RNA sequencing and single-cell & single-nucleus sequencing in the same tissue. By improving these three technical solutions, the present invention achieves the simultaneous detection of exosomal small RNA, single-cell, and single-nucleus transcriptomes in the same intrahepatic bile duct tissue, which has broad application prospects, including but not limited to analyzing the operating mechanisms of regulatory networks behind life phenomena.

[0008] The term "simultaneously" in this invention refers to the ability to obtain exosomes, single-cell suspensions, and single-cell nuclei suspensions from the same tissue, regardless of the order in which they are obtained. Because nuclear lysis solutions can damage cells, the present invention prioritizes the preparation of single-cell suspensions over the preparation of single-cell nuclei suspensions.

[0009] The present invention's "exo+snRNA-seq & scRNA-seq" refers to the simultaneous preparation of exosomes, single-cell suspensions, and single-cell nucleus suspensions in the same intrahepatic bile duct tissue, and the simultaneous performance of exosome smallRNA sequencing, single-cell & single-cell nucleus sequencing.

[0010] At present, there is no technology that can simultaneously prepare exosome suspension, single cell suspension and single cell nucleus suspension in the same tissue. How to prepare three suspensions simultaneously in the same tissue is the main problem that needs to be solved at present. The present invention creatively uses compound enzymes (collagenase II solution, collagenase IV solution, hyaluronidase solution, pancreatic enzyme solution) to efficiently enzymatically hydrolyze tissue in a short time (15 minutes), thereby achieving the purpose of being able to simultaneously prepare exosomes, single cell suspension and single cell nucleus suspension in the same tissue, and greatly reducing the impact of tissue enzymatic hydrolysis on subsequent cell nucleus preparation and exosome extraction. The exo+scRNA-seq&snRNA-seq process proposed in the present invention realizes the simultaneous sequencing of single cell (nuclear) transcriptome and exosome smallRNA in the same sample. The present invention can understand how complete cell clusters from the same tissue source communicate through exosomes, and what role they play in tumor development, microenvironment remodeling, metastasis, drug resistance, etc. It breaks through the barriers of existing technical methods, fills the current technological gap, and provides a more systematic, in-depth principle and more precise mechanism for the present invention to reveal the cell interaction network.

[0011] The present invention provides a method for simultaneously preparing exosomes, single cell suspension, and single cell nucleus suspension based on the same intrahepatic bile duct tissue sample, comprising the following steps:

[0012] (1) Sample preparation:

[0013] Obtain human intrahepatic bile duct tissue, wash it, mince it, and transfer the minced tissue pieces into a centrifuge tube;

[0014] (2) Enzymatic hydrolysis:

[0015] Add culture medium, collagenase II, collagenase IV, and hyaluronidase solution for digestion and enzymatic hydrolysis at 37°C. After enzymatic hydrolysis at 37°C, add trypsin solution, mix well, and let stand at room temperature;

[0016] (3) Separation:

[0017] After standing at room temperature, a first supernatant and a first settled tissue pellet are obtained, and the collected first supernatant is subjected to a first centrifugation to obtain a second supernatant and a second cell pellet;

[0018] (IV) Exosome preparation:

[0019] The second supernatant was centrifuged a second time to obtain a third supernatant and a third cell pellet. After centrifugation, the third supernatant was sieved and the third filtrate was subjected to exosome purification using the Invitrogen-Total Exosome Isolation Reagent from cell culture media kit. Exosome purification involves mixing the sieved third supernatant with Invitrogen reagents and incubating them. After incubation, the mixture was centrifuged, the pellet was recovered, and the pellet was resuspended in PBS buffer to obtain an exosome suspension.

[0020] (V) Preparation of suspension:

[0021] Preparation of single cell suspension:

[0022] Take the second cell pellet and the third cell pellet in the above step (iii) and resuspend them in culture medium respectively, transfer them to the same centrifuge tube, and perform sieving. After the sieving process, perform the first centrifugation, discard the supernatant after the centrifugation, add red blood cell lysis solution to the cell pellet, mix by pipetting, and let it stand at room temperature. After the red blood cell lysis is completed, perform the second centrifugation, discard the supernatant after the centrifugation, add dead cell reagent (magnetic beads) to resuspend the cell pellet, incubate for 15 minutes, rinse the LS column with 1× building buffer, pass the cell mixture through the column, centrifuge for the third time, discard the supernatant after the centrifugation, add pre-cooled culture medium to perform the fourth centrifugation washing process on the cell pellet, discard the supernatant after the centrifugation, add pre-cooled culture medium to resuspend the cell pellet to obtain a single cell suspension.

[0023] Preparation of single cell nucleus suspension:

[0024] The first precipitated tissue pellet in the above step (iii) was washed with PBS, the supernatant was discarded after the first centrifugal wash, and PBS was added for washing again. After washing, Lysis Buffer was added to resuspend the tissue pellet and start lysis treatment. The entire process was incubated on ice. After the lysis was completed, the tissue pellet was sieved and the filtrate was collected after sieving. After the second centrifugation, the supernatant was discarded, STWash Buffer was added to resuspend the cell nuclear pellet, and after the third centrifugal wash, the supernatant was discarded and the cell nuclear pellet was resuspended with PBS containing 1% BSA to obtain a single cell nucleus suspension.

[0025] Preferably, the tissue sample in the present invention is human intrahepatic bile duct carcinoma tissue.

[0026] In a specific embodiment, the method of the present invention comprises the following steps:

[0027] I. Prepare Reagents

[0028] 1.1 Culture medium, trypsin solution, collagenase II solution, collagenase IV solution, hyaluronidase solution, pre-chilled PBS solution, lysis solution, and dead cell removal reagent (Dead Cell Removal Microbeads, Binding Buffer).

[0029] 2.1 Prepare Lysis Buffer, ST Wash Buffer, and final buffer, and pre-cool the prepared solutions on crushed ice.

[0030] 3.1 Prepare Invitrogen-exosomes purification reagent, Exosomal RNA Isolation Kit

[0031] II. Preparation

[0032] 1.2 Sample preparation

[0033] Fresh human intrahepatic bile duct tissue was obtained and placed in a culture dish. The culture dish was placed on an ice plate. The tissue was washed with pre-cooled culture medium to clean the blood on the tissue surface and then the washing solution was added.

[0034] 1.3 Tissue fragmentation

[0035] Use sterile surgical scissors to break up the tissue. Stop when the tissue is cut into a paste and transfer the minced tissue pieces in the culture dish to a new centrifuge tube.

[0036] 1.4 Tissue enzymatic hydrolysis

[0037] After adding pre-chilled culture medium to the centrifuge tube, add collagenase II, collagenase IV, and hyaluronidase solutions. Mix thoroughly by pipetting with a disposable pipette. Place the centrifuge tube in a hybridization oven for enzymatic digestion at 37°C. After enzymatic digestion, add trypsin solution to the centrifuge tube, mix thoroughly by inverting, and let it stand at room temperature.

[0038] 1.5 End of enzymatic hydrolysis

[0039] After enzymatic hydrolysis, use a disposable pipette to mix thoroughly by pipetting. After mixing and pipetting, let the mixture stand for a while. Once the tissue fragments have completely settled, remove the supernatant with a pipette and transfer it to a new 15mL centrifuge tube for later use. Recover the remaining tissue pellet for subsequent preparation of a single-cell nucleus suspension. Centrifuge the supernatant for the first time. After centrifugation, transfer the first supernatant to a new centrifuge tube and centrifuge it a second time. Reserve the first cell pellet for later use. After the second centrifugation, collect the second supernatant for subsequent exosome preparation. Resuspend the first and second cell pellets in culture medium and transfer them together to a new centrifuge tube for subsequent preparation of a single-cell suspension.

[0040] Prepare a single-cell suspension by following steps 1.6-1.9:

[0041] 1.6 Sieving

[0042] The mixture of the first and second cell pellets resuspended in the culture medium in step 1.5 was added dropwise to a 40 μm mesh and filtered. After centrifugation, the supernatant was discarded.

[0043] 1.7 Red crack

[0044] After adding red blood cell lysis buffer and mixing with the cell pellet, let it stand, centrifuge after the standing period, and discard the supernatant after the centrifugation.

[0045] 1.8 Removal of dead cells

[0046] Add Dead Cell Removal MicroBeads solution, incubate, rinse the LS column with building buffer, and then pass the column. After the incubation period, centrifuge and discard the supernatant.

[0047] 1.9 Washing and microscopic examination

[0048] Add pre-chilled culture medium to the cell pellet and centrifuge at 4°C to wash. Discard the supernatant after centrifugation. Resuspend the cell pellet in pre-chilled culture medium and examine under a microscope to determine the final total cell count, cell viability, and debris percentage.

[0049] Prepare a single cell nucleus suspension according to steps 2.2-2.7:

[0050] 2.2 Washing tissue

[0051] Add PBS to the enzymatically hydrolyzed tissue pellet retained in step 1.5 above, wash the tissue pellet, and discard the supernatant after centrifugation.

[0052] 2.3 Cracking

[0053] Add Lysis Buffer to the tissue pellet washed in 2.2 above. Mix thoroughly by pipetting with a disposable pipette. Incubate on crushed ice for a desired period of time. Examine under a microscope after lysis is complete.

[0054] 2.4 Screening

[0055] The cell nucleus suspension was sieved, and the filtrate was collected into a new centrifuge tube after sieving, and the supernatant was discarded after centrifugation.

[0056] 2.5 Washing and purification

[0057] Add STWash Buffer to resuspend the nuclear pellet obtained by centrifugation in 2.4. After centrifugation, discard the supernatant. Repeat the wash.

[0058] 2.6 Quality Inspection

[0059] Add the final buffer to the cell nuclear pellet obtained in 2.5 above, resuspend the cell nuclear pellet, and finally obtain the cell nuclear suspension used for the machine.

[0060] 2.7 Quality Inspection

[0061] Take the cell nucleus suspension obtained in step 2.6 above for microscopic examination, dilute it after microscopic examination, and determine the final cell nucleus concentration, cell nucleus fragment ratio, and cell nucleus agglomeration ratio for the instrument.

[0062] 2.8 Sequencing

[0063] Single-cell sequencing was performed according to the 10x Genomics instructions.

[0064] Prepare purified exosomes according to steps 3.2-3.4:

[0065] 3.2 Defragmentation

[0066] The supernatant from the second centrifugation in step 1.5 above was centrifuged and after the centrifugation was completed, the supernatant was collected into a new centrifuge tube.

[0067] 3.3 Incubation

[0068] The supernatant after centrifugation in 3.2 was mixed with Invitrogen reagent and incubated at 4°C.

[0069] 3.4 Recycling

[0070] The mixed solution after incubation in the above 3.3 was centrifuged, the precipitate was recovered after centrifugation, and the precipitate was resuspended in a buffer solution.

[0071] 3.5 RNA extraction and quantification

[0072] RNA was extracted from the exosomes obtained in step 3.4 using a kit. All procedures were performed according to the kit instructions. The obtained RNA was quantified using a NanoDrop 2000 to determine the amount of RNA obtained.

[0073] In a specific embodiment,

[0074] In step 1.1, the culture medium refers to RPMI1640 medium supplemented with 1% BSA. The RPMI1640 medium is manufactured by Corning, with the catalog number CGR-10-040-CV. The BSA is manufactured by MACS, with the catalog number 130091376.

[0075] In step 1.1, the pre-cooling temperature of the pre-cooled PBS is 0-5°C; preferably, it is pre-cooled at 4°C.

[0076] In step 1.1, the PBS manufacturer is Gibco, and the product number is 10010-031.

[0077] In step 1.1, the pancreatic enzyme solution is prepared by dissolving lyophilized pancreatic enzyme powder in PBS to prepare a pancreatic enzyme solution with a concentration of 2.5% (m / v), filtering the solution through a 0.22 μm filter, and storing the solution at -20°C for later use.

[0078] In step 1.1, the collagenase II solution is prepared by dissolving lyophilized collagenase powder in HBSS solution containing calcium and magnesium ions to prepare a collagenase solution with a concentration of 1% (m / v), filtering the solution using a 0.22 μm filter, and storing the solution at -20°C for later use.

[0079] In step 1.1, the collagenase IV solution is prepared by dissolving lyophilized collagenase powder in HBSS solution containing calcium and magnesium ions to prepare a collagenase solution with a concentration of 1% (m / v), filtering the solution using a 0.22 μm filter, and storing the solution at -20°C for later use.

[0080] In step 1.1, the manufacturer of the pancreatic enzyme dry powder is Solebow, and the product number is T8150-10g.

[0081] In step 1.1, the collagenase II dry powder is produced by Gibco, with the product number 17101-015.

[0082] In step 1.1, the collagenase IV dry powder is produced by Gibco, with the product number 17104-019.

[0083] In step 1.1, the hyaluronidase dry powder is produced by Sigma and its product number is H3506.

[0084] In step 1.1, the HBSS manufacturer is Gibco, and the product number is 14025-076.

[0085] In step 1.1, the red lysis solution is manufactured by Shanghai Sangon Biotechnology Co., Ltd., and the product number is B541001-0100.

[0086] In step 1.1, the dead cell removal reagents (Dead Cell Removal MicroBeads, Binding Buffer) are manufactured by Miltenyi Blotec with a catalog number of 130-109-398 and a Binding Buffer with a catalog number of 130-090-101.

[0087] In step 1.2, the fresh weight of the fresh human intrahepatic bile duct tissue is 500-800 mg; preferably, 600 mg.

[0088] In step 1.2, the purpose of washing the tissue with culture medium is to fully clean the blood remaining on the human intrahepatic bile duct tissue sample to reduce the proportion of red blood cells in subsequent sample processing.

[0089] In step 1.2, the sample is washed 1 to 4 times; preferably, 3 times.

[0090] In step 1.3, the centrifuge tube is a 15 mL centrifuge tube manufactured by Corning with a product number of 430790.

[0091] In step 1.3, the scissors are sterile ophthalmic scissors that have been sterilized at high temperature.

[0092] In step 1.4, the final concentration of collagenase II is 0.5% (v / v).

[0093] In step 1.4, the final concentration of collagenase IV is 0.3% (v / v).

[0094] In step 1.4, the final concentration of the hyaluronidase is 0.1% (v / v).

[0095] In step 1.4, the digestion and enzymatic hydrolysis are carried out in the hybridization oven at 37° C. for 10 to 20 minutes, preferably 15 minutes.

[0096] In step 1.4, the final concentration of trypsin is 0.2% (v / v).

[0097] In step 1.4, the standing time at room temperature is 3 to 6 minutes; preferably, 4 minutes.

[0098] In step 1.4, the pre-cooled culture medium refers to adding 1% BSA to RPMI1640 culture medium, wherein the pre-cooling temperature is 4°C.

[0099] In step 1.5, the enzymatic hydrolysis is completed after the digestion is completed in the 37°C hybridization oven and then the trypsin is used to stand at room temperature.

[0100] In step 1.5, the purpose of transferring the supernatant to a new 15m centrifuge tube is to prepare exosomes and single cell suspension.

[0101] In step 1.5, the purpose of transferring the remaining tissue pellet to a new 15 mL centrifuge tube is to prepare a cell nucleus suspension.

[0102] In step 1.5, the first centrifugation conditions are 2000-3000×g, 5-10 min, 4-6°C; preferably, 3000×g, 4°C, 10 min.

[0103] In step 1.5, the first supernatant is transferred to a new centrifuge tube, which is a 1.5 mL centrifuge tube manufactured by CNW with a product number of ABEQ-5615000-500.

[0104] In step 1.5, the second centrifugation conditions are 13000×g to 16000×g, 5 to 10 min, and 4 to 6°C; preferably, 16000×g, 4°C, and 10 min.

[0105] In step 1.5, the second centrifugation is to remove impurities and debris.

[0106] In step 1.5, the purpose of resuspending the first cell pellet and the second cell pellet in culture medium and then transferring them to a new 15 mL centrifuge tube for standby use is to prepare a single cell suspension.

[0107] In step 1.6, the volume of the culture medium added for re-suspension is 4 to 8 mL; preferably, it is 7 mL.

[0108] In step 1.6, the centrifugation conditions are 500-800×g, 5-10 min, 4-6°C; preferably, 500×g, 4°C, 10 min.

[0109] In step 1.7, the volume of the red blood cell lysis solution is 6 to 10 mL; preferably, 8 mL.

[0110] In step 1.7, the standing time is 4 to 8 minutes, preferably 5 minutes.

[0111] In step 1.7, the centrifugation conditions are 500-800×g, 5-10 min, 4-6°C; preferably, 500×g, 4°C, 5 min.

[0112] In step 1.8, the incubation condition is 15 minutes at room temperature.

[0113] In step 1.8, the centrifugation conditions are 300-500×g, 5-10 min, 4-6°C; preferably, 400×g, 4°C, 5 min.

[0114] In step 1.8, the building buffer is 20× building buffer, which is diluted with enzyme-free water to prepare 1× building buffer.

[0115] In step 1.8, rinsing the LS column with building buffer refers to slowly adding 3 mL of 1× building buffer to the LS column using a pipette.

[0116] In step 1.8, the LS column is manufactured by Miltenyi Blotec, with a product number of 130-042-401.

[0117] In step 1.9, the volume of the culture medium added for washing is 4 to 8 mL; preferably, 6 mL.

[0118] In step 1.9, the centrifugation conditions are 100-500×g for 5-10 min; preferably, 300×g, 4°C, for 7 min.

[0119] In step 1.9, the washing times are 1 to 3 times; preferably, 2 times.

[0120] In step 1.9, the pre-cooled culture medium refers to adding 1% BSA to RPMI1640 culture medium, wherein the pre-cooling temperature is 4°C.

[0121] In step 1.9, the volume of culture medium added is 0.5 to 2 mL; preferably, 1.5 mL.

[0122] In step 1.9, the microscopic examination refers to taking 9 μL of the single cell suspension and mixing it with 1 μL of 0.4% trypan blue solution, and then performing microscopic examination.

[0123] In step 1.9, the trypan blue solution is produced by Thermo Fisher Scientific with a product number of T10282.

[0124] In step 1.9, the total number of cells in the single-cell suspension is 3 million, the cell viability is 96%, and the debris ratio is 6%.

[0125] In step 2.1, the lysis buffer includes the following components (final concentration): 50 mM Tris-HCl (pH = 7.5), 1 mM CaCl2, 5 mM NaCl, 0.3% to 0.4% (v / v) NP40, 0.3% to 0.4% (v / v) Tween 20, and 0.4 U / μL RNase Inhibitor.

[0126] In step 2.1, the cell nucleus washing buffer contains the following components (final concentration): 50 mM Tris-HCl (pH = 7.5), 1 mM CaCl2, 5 mM NaCl, 0.4 U / μL RNase Inhibitor (RNase inhibitor), and 1% to 2% (v / v) BSA.

[0127] In step 2.1, the final cell nucleus buffer comprises the following components (final concentrations): 9% PBS, 1% to 2% (v / v) BSA.

[0128] In step 2.1, the main component of the final cell nucleus buffer solution is BSA, with a final concentration of 1% to 2% (v / v); preferably, 1% (v / v).

[0129] In step 2.1, the components were purchased from Tris-HCl (Invitrogen; 15567027), CaCl2 (Sigma-Aldrich; 21115-100ML), NaCl (Thermo Fisher Scientific; AM9760G), NP40 (ThermoScientific TM ; 28324), RNase inhibitor (Enzymatics & QIAGEN; Y9240L) BSA (MACS, 130091376).

[0130] In step 2.1, the ice refers to the crushed ice produced by the ice maker, which is placed in a foam box of appropriate size, and the centrifuge tube is inserted into the crushed ice.

[0131] In step 2.2, the volume of PBS is 10-15 mL; preferably, 14 mL.

[0132] In step 2.2, the tissue is washed 1 to 3 times; preferably, 3 times.

[0133] In step 2.2, the purpose of washing the tissue is to clean the residual enzyme solution in the tissue to avoid affecting the subsequent preparation of cell nuclei.

[0134] In step 2.2, the centrifugation conditions are 500g-1000×g for 5-10 min; preferably, 800×g, 4°C, for 10 min.

[0135] In step 2.3, the volume of the lysis buffer is 2 to 5 mL; preferably, 4 mL.

[0136] In step 2.3, the incubation and lysis on ice for a certain time is 5 to 10 minutes; preferably, 8 minutes.

[0137] In step 2.3, the microscopic examination was performed using 0.4% trypan blue (v / v): 1 μL of trypan blue solution was mixed with 9 μL of cell nucleus suspension for microscopic examination.

[0138] In step 2.4, the pore size of the cell sieve is 40 μm.

[0139] In step 2.4, the cell screen was purchased from BD falcon, catalog number 352340.

[0140] In step 2.4, the new centrifuge tube is a 15 mL centrifuge tube manufactured by Corning with a product number of 430790.

[0141] In step 2.4, the centrifuge conditions are 800-1000×g, 4-6°C, and centrifugation for 5-15 minutes; preferably, 800×g, 4°C, and centrifugation for 8 minutes.

[0142] In step 2.5, the volume of the STWash Buffer is 5 to 8 mL; preferably, 6 mL.

[0143] In step 2.5, the centrifugation conditions for washing are 500-1000×g and 4°C for 5-10 minutes; preferably, 800×g and 4°C for 10 minutes.

[0144] In step 2.5, the washing times are 1 to 3 times; preferably, 2 times.

[0145] In step 2.6, the final volume of the buffer is 1-2 mL; preferably, 1.5 mL.

[0146] In step 2.7, the post-microscopic dilution is to dilute the cell nuclei suspension to a concentration of 1200-1600 cells / μl using the final buffer.

[0147] In step 2.7, the microscopic examination was performed using 0.4% trypan blue (v / v): 1 μL of trypan blue solution was mixed with 9 μL of cell nucleus suspension for microscopic examination.

[0148] In step 2.7, the trypan blue solution is manufactured by Thermo Fisher Scientific with a product number of T10282.

[0149] In step 3.1, the exosome purification reagent is manufactured by Invitrogen with a catalog number of 4478359.

[0150] In step 3.1, the Exosomal RNA Isolation Kit is manufactured by Norgen Biotek and the product number is NGB-58000.

[0151] In step 3.2, the centrifugation is to remove debris and impurities again.

[0152] In step 3.2, the centrifugation conditions are 16,000×g and 4°C for 5 to 10 minutes; preferably, 16,000×g and 4°C for 10 minutes.

[0153] In step 3.2, the new centrifuge tube is a 15 mL centrifuge tube manufactured by Corning with a product number of 430790.

[0154] In step 3.3, the incubation temperature is 2-8°C; preferably, 4°C.

[0155] In step 3.3, the incubation time is 8 to 12 hours; preferably, 10 hours.

[0156] In step 3.3, the centrifugation conditions are 10,000×g, 4° C., and 60 minutes.

[0157] In step 3.4, the buffer solution is PBS, manufactured by Gibco, and the product number is 10010-031.

[0158] In step 3.5, the small RNA in the exosomes is extracted, a library is constructed for the small RNA, and NGS sequencing is performed.

[0159] The present invention also provides exosomes, single cell suspension and single cell nucleus suspension obtained by the method.

[0160] The exosome yield was high, the total exosome RNA content was 300 ng, and the exosome concentration (particles / mL) was 1.06E+10. Furthermore, the exosome total RNA was less contaminated by impurities such as proteins (using 260 / 280 as a quality control indicator for protein and other impurity contamination), with a 260 / 280 ratio of 2.01.

[0161] The single-cell suspension has a high yield, meeting the 10x Genomics standards. The total cell count of the prepared single-cell suspension can reach 98W, and the single-cell suspension has few impurities, with a cell suspension fragment rate of 4% and a clumping rate of 4%.

[0162] The single-cell nucleus suspension has a high yield, meeting the 10x Genomics standards. The total number of nuclei in the prepared single-cell nucleus suspension can reach 220W, and the single-cell nucleus suspension has few impurities, with a fragmentation rate of 5% and a clumping rate of 5%.

[0163] The present invention also proposes a reagent / kit that can be used to simultaneously prepare single-cell suspension and single-cell nucleus suspension in the same human intrahepatic bile duct tissue sample, which includes but is not limited to cell enzymatic hydrolysis solution, cell nucleus lysis solution, and cell nucleus washing buffer.

[0164] Wherein, the cell enzymatic solution is 0.5% collagenase II (v / v) solution, 0.3% collagenase IV solution, 0.1% hyaluronidase solution, and 0.2% (v / v) trypsin;

[0165] The cell nuclear lysis solution is the lysis buffer comprising the following components (final concentrations): 50 mM Tris-HCl (pH = 7.5), 1 mM CaCl2, 5 mM NaCl, 0.3% to 0.4% (v / v) NP40, 0.3% to 0.4% (v / v) Tween 20, 0.4 U / μL RNase Inhibitor;

[0166] The cell nucleus washing buffer contains the following components (final concentration): 50 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 0.4 U / μL RNase Inhibitor, and 1% to 2% (v / v) BSA.

[0167] The present invention also proposes the application of the reagent / kit, which can be used to efficiently, quickly, and stably obtain single-cell suspensions and single-cell nucleus suspensions with a large total amount and low fragmentation and agglomeration ratios, and can be used in the preparation of scRNA-seq and snRNA-seq simultaneously in the same human intrahepatic bile duct tissue.

[0168] The kit provided by the present invention has little impact on the operation of different personnel when in use, has good repeatability, is ready for use after disassembly, does not require complicated procedures, and is easy to operate, gentle, and efficiently obtains high-quality single-cell suspensions.

[0169] Compared with the existing technology, the beneficial effects of the present invention also include: the present invention fills the current technical gap. At present, there is no technology for preparing single-cell suspension, single-cell nuclear suspension and exosomes in the same tissue or the same intrahepatic bile duct tissue at the same time. The technical solution provided by the present invention solves this problem and fills the technical gap. The technical solution provided by the present invention can obtain more meaningful data in the same sample and mine deeper biological information. The present invention innovatively and improvedly combines the three technologies of exosome preparation, single-cell suspension preparation and single-cell nuclear suspension preparation, breaking through the existing technical barriers, obtaining a more comprehensive cell type in the sample, and truly analyzing the network mechanism of cell interaction behind life phenomena (both understanding the function of individual cells and how cells transmit signals), thereby enriching the research data and providing a basis for mining deeper biological mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1 It is a schematic diagram of the microscopic examination results of single cell suspension of intrahepatic bile duct in the method of the present invention.

[0171] Figure 2 It is a schematic diagram of the microscopic examination results of single cell nuclear suspension of intrahepatic bile duct in the method of the present invention.

[0172] Figure 3 It is a schematic diagram of the quality inspection results of intrahepatic bile duct cell RNA in the method of the present invention.

[0173] Figure 4 It is a schematic diagram of the quality inspection results of intrahepatic bile duct cell nuclear RNA in the method of the present invention.

[0174] Figure 5 This is a schematic diagram of the TEM detection results of exosomes isolated from intrahepatic bile duct tissue in the method of the present invention.

[0175] Figure 6 This is a schematic diagram of the NTA detection results of exosomes isolated from intrahepatic bile duct tissue in the method of the present invention.

[0176] Figure 7 It is a schematic diagram of the TSNE atlas results of intrahepatic bile duct cells and cell nuclei in the method of the present invention.

[0177] Figure 8 This is a schematic diagram of the present invention's simultaneous preparation of exosomes, single cell suspension, and single cell nucleus suspension based on the same intrahepatic bile duct tissue sample, as well as the exo+scRNA-seq+snRNA-seq process.

[0178] Figure 9 Schematic diagram of the kit for preparing single cell suspension and cell nucleus suspension simultaneously from the same intrahepatic bile duct tissue sample in the method of the present invention. DETAILED DESCRIPTION

[0179] The invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0180] Example 1

[0181] In this example, exosomes, single-cell suspensions, and single-cell nuclear suspensions were prepared simultaneously from the same intrahepatic bile duct, and exosome small RNA sequencing and single-cell (nuclear) sequencing were performed, respectively.

[0182] 1.1 Prepare reagents

[0183] Culture medium, trypsin solution, collagenase II solution, collagenase IV solution, hyaluronidase solution, pre-cooled PBS solution, lysate, dead cell removal reagent (Dead Cell Removal MicroBeads, Binding Buffer

[0184] 1.2 Sample preparation

[0185] 600 mg of fresh intrahepatic bile duct tissue was obtained, washed with PBS, wiped dry, and placed in a culture dish. The culture dish was placed on an ice plate, and the tissue was washed with pre-cooled culture medium. After completion, the washing solution was discarded.

[0186] 1.3 Tissue fragmentation

[0187] Use sterile scissors to mince the intrahepatic bile duct tissue. Stop when the tissue is chopped into a paste and transfer the minced tissue pieces in the culture dish to a new 15 mL centrifuge tube.

[0188] 1.4 Tissue enzymatic hydrolysis

[0189] Add 8 mL of pre-chilled culture medium, a final concentration of 0.5% collagenase II (v / v), 0.3% collagenase IV, and 0.1% hyaluronidase (v / v). Mix by inversion and allow to digest in a hybridization oven at 37°C for 15 minutes. After digestion, add 0.2% (v / v) pancreatin solution to the centrifuge tube, mix by inversion, and let stand at room temperature for 4 minutes.

[0190] 1.5 End of enzymatic hydrolysis

[0191] After standing at room temperature, wait for the tissue blocks to completely settle, and use a pipette to remove the supernatant and transfer it to a new 15mL centrifuge tube. Keep the tissue pellet at the bottom for the preparation of a single cell nucleus suspension. Centrifuge the supernatant for the first time at 3000×g and 4°C for 10 minutes. After the centrifugation is completed, transfer the supernatant to a new centrifuge tube and centrifuge for a second time at 16000×g and 4°C for 10 minutes. The first cell pellet is retained for later use. After the second centrifugation is completed, collect the supernatant from the second centrifugation for subsequent exosome preparation. Resuspend the first and second cell pellets in 4mL of culture medium and transfer them together to a new centrifuge tube for the subsequent preparation of a single cell suspension.

[0192] Prepare a single-cell suspension as follows 1.6-1.9:

[0193] 1.6 Sieving

[0194] The cell pellet resuspended in 1.5 was pipetted through a 40 μm mesh filter into a new 15 mL centrifuge tube. After sieving, the pellet was centrifuged at 500 × g and 4°C for 10 min. The supernatant was discarded.

[0195] 1.7 Lysis of red blood cells

[0196] Add 8 mL of red blood cell lysis buffer to the cell pellet, mix thoroughly by pipetting, and let stand at room temperature for 5 minutes. After lysis, centrifuge at 500 × g, 4°C, for 5 minutes. Discard the supernatant.

[0197] 1.8 Removal of dead cells

[0198] Add 500 μL of Dead Cell Removal MicroBeads solution to the cell pellet and incubate at room temperature for 15 minutes. Rinse the LS column with 1× building buffer 1 minute before the end of the incubation period. After the column run, centrifuge at 400 × g and 4°C for 5 minutes. Discard the supernatant.

[0199] 1.9 Washing and microscopic examination

[0200] Add 6 mL of culture medium to the cell pellet and resuspend it. Centrifuge at 300 × g, 4°C, for 7 min. Discard the supernatant. After washing, resuspend the cell pellet in 1 mL of pre-chilled culture medium and examine it under a microscope using 0.4% trypan blue (ThermoFisher Scientific, T10282). Mix 1 μL of trypan blue solution with 9 μL of cell suspension and examine the mixture under a microscope.

[0201] 2.1 Prepare reagents

[0202] Prepare lysis buffer, wash buffer and final buffer separately and pre-cool the prepared solutions.

[0203] The lysis buffer includes the following components (final concentrations): 50 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 0.3% (v / v) NP40, 0.3% (v / v) Tween 20, and 0.4 U / μL RNase Inhibitor.

[0204] The washing buffer contained the following components (final concentrations): 50 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 0.4 U / μL RNase Inhibitor, and 1% (v / v) BSA.

[0205] The final buffer contains the following components (final concentrations): 9% PBS, 1% (v / v) BSA, and 0.4 U / μL RNase Inhibitor.

[0206] Prepare single cell nuclei suspension as follows 2.2-2.6:

[0207] 2.2 Tissue Precipitate Washing

[0208] Wash the remaining tissues described in 1.5 three times by adding 14 mL of PBS, centrifuge at 800 × g and 4°C for 10 min, and discard the supernatant after centrifugation.

[0209] 2.3 Lysis of tissue

[0210] Add 4 mL of cell lysis buffer to the washed tissue pellet and incubate on ice for 8 min.

[0211] 2.4 Screening

[0212] After the lysis is completed, the cell suspension is filtered using a 40 μm cell sieve and transferred to a new 15 mL centrifuge tube. The filtrate is centrifuged at 800 × g and 4° C. for 8 min, and the supernatant is discarded.

[0213] 2.5 Washing

[0214] Add 6 mL of washing buffer to the nuclear pellet, centrifuge at 800 × g and 4°C for 8 min, and discard the supernatant after centrifugation.

[0215] 2.6 Washing

[0216] Add 6 mL of wash buffer to the nuclear pellet from the previous step and centrifuge at 800 × g, 4°C for 5 min. Discard the supernatant. Add 1.5 mL of final buffer to the final nuclear pellet and resuspend it to obtain the nuclear suspension for use in the instrument.

[0217] 2.7 Microscopic examination

[0218] 0.4% trypan blue (Thermo Fisher Scientific, T10282) was used for microscopic examination: 1 μL of trypan blue solution was mixed with 9 μL of cell nucleus suspension and then examined under a microscopic microscope.

[0219] 3.1 Prepare reagents

[0220] Invitrogen-exosomes purification reagent, Exosomal RNA Isolation Kit, PBS

[0221] Prepare single cell nuclei suspension as follows 3.2-3.4

[0222] 3.2 Defragmentation

[0223] Centrifuge the supernatant from the second centrifugation in step 1.5 above at 16,000 × g, 4°C, for 10 min. After centrifugation, collect the supernatant into a new centrifuge tube.

[0224] 3.3 Incubation

[0225] The supernatant after centrifugation in 3.2 was mixed with Invitrogen reagent and incubated at 4°C for 10 h.

[0226] 3.4 Recycling

[0227] The mixture after incubation in 3.3 was centrifuged at 10,000 × g and 4°C for 60 minutes. The precipitate was recovered after centrifugation and resuspended in PBS buffer.

[0228] 3.5 RNA extraction and quantification

[0229] RNA was extracted from the exosomes obtained in step 3.4 using a kit. All procedures were performed according to the kit instructions (Exosomal RNA Isolation Kit). The obtained RNA was quantified using a NanoDrop 2000 to determine the amount of RNA obtained.

[0230] Results and analysis:

[0231] The above experimental results show that the method of the present invention allows the simultaneous preparation of exosomes, single-cell suspensions, and single-nucleus suspensions from the same intrahepatic bile duct tissue. After preparation, the single-cell suspensions and single-nucleus suspensions, after microscopic examination with trypan blue staining, showed that the cell and nucleus concentrations, debris and impurity ratios, and clumping ratios all met the requirements for single-cell sequencing by 10x Genomics. Furthermore, the small RNA isolated from the exosomes and the resulting libraries also met the requirements for NGS sequencing (see Table 1).

[0232] Example 2

[0233] In this example, exosomes, single-cell suspensions, and single-cell nuclear suspensions were prepared simultaneously from the same intrahepatic bile duct, and exosome small RNA sequencing and single-cell (nuclear) sequencing were performed, respectively.

[0234] 1.1 Prepare reagents

[0235] Culture medium, trypsin solution, collagenase II solution, collagenase IV solution, hyaluronidase solution, pre-cooled PBS solution, lysate, dead cell removal reagent (Dead Cell Removal MicroBeads, Binding Buffer

[0236] 1.2 Sample preparation

[0237] 600 mg of fresh intrahepatic bile duct tissue was obtained, washed with PBS, wiped dry, and placed in a culture dish. The culture dish was placed on an ice plate, and the tissue was washed with pre-cooled culture medium. After completion, the washing solution was discarded.

[0238] 1.3 Tissue fragmentation

[0239] Use sterile scissors to mince the intrahepatic bile duct tissue. Stop when the tissue is chopped into a paste and transfer the minced tissue pieces in the culture dish to a new 15 mL centrifuge tube.

[0240] 1.4 Tissue enzymatic hydrolysis

[0241] Add 8 mL of pre-chilled culture medium, a final concentration of 0.5% collagenase II (v / v), 0.3% collagenase IV, and 0.1% hyaluronidase (v / v). Mix by inversion and allow to digest in a hybridization oven at 37°C for 15 minutes. After digestion, add 0.2% (v / v) pancreatin solution to the centrifuge tube, mix by inversion, and let stand at room temperature for 4 minutes.

[0242] 1.5 End of enzymatic hydrolysis

[0243] After standing at room temperature, wait for the tissue blocks to completely settle, and use a pipette to remove the supernatant and transfer it to a new 15mL centrifuge tube. Keep the tissue pellet at the bottom for the preparation of a single cell nucleus suspension. Centrifuge the supernatant for the first time at 3000×g and 4°C for 10 minutes. After the centrifugation is completed, transfer the supernatant to a new centrifuge tube and centrifuge for a second time at 16000×g and 4°C for 10 minutes. The first cell pellet is retained for later use. After the second centrifugation is completed, collect the supernatant from the second centrifugation for subsequent exosome preparation. Resuspend the first and second cell pellets in 4mL of culture medium and transfer them together to a new centrifuge tube for the subsequent preparation of a single cell suspension.

[0244] Prepare a single-cell suspension as follows 1.6-1.9:

[0245] 1.6 Sieving

[0246] The cell pellet resuspended in 1.5 was pipetted through a 40 μm mesh filter into a new 15 mL centrifuge tube. After sieving, the pellet was centrifuged at 500 × g and 4°C for 10 min. The supernatant was discarded.

[0247] 1.7 Lysis of red blood cells

[0248] Add 8 mL of red blood cell lysis buffer to the cell pellet, mix thoroughly by pipetting, and let stand at room temperature for 5 minutes. After lysis, centrifuge at 500 × g, 4°C, for 5 minutes. Discard the supernatant.

[0249] 1.8 Removal of dead cells

[0250] Add 500 μL of Dead Cell Removal MicroBeads solution to the cell pellet and incubate at room temperature for 15 minutes. Rinse the LS column with 1× building buffer 1 minute before the end of the incubation period. After the column run, centrifuge at 400 × g and 4°C for 5 minutes. Discard the supernatant.

[0251] 1.9 Washing and microscopic examination

[0252] Add 6 mL of culture medium to the cell pellet and resuspend it. Centrifuge at 300 × g, 4°C, for 7 min. Discard the supernatant. After washing, resuspend the cell pellet in 1 mL of pre-chilled culture medium and examine it under a microscope using 0.4% trypan blue (ThermoFisher Scientific, T10282). Mix 1 μL of trypan blue solution with 9 μL of cell suspension and examine the mixture under a microscope.

[0253] 2.1 Prepare reagents

[0254] Prepare lysis buffer, wash buffer and final buffer separately and pre-cool the prepared solutions.

[0255] The lysis buffer includes the following components (final concentrations): 50 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 0.4% (v / v) NP40, 0.4% (v / v) Tween 20, and 0.4 U / μL RNase Inhibitor.

[0256] The washing buffer contained the following components (final concentrations): 50 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 0.4 U / μL RNase Inhibitor, and 1% (v / v) BSA.

[0257] The final buffer contains the following components (final concentrations): 9% PBS, 1% (v / v) BSA, and 0.4 U / μL RNase Inhibitor.

[0258] Prepare single cell nuclei suspension as follows 2.2-2.6:

[0259] 2.2 Tissue Precipitate Washing

[0260] Wash the remaining tissues described in 1.5 three times by adding 14 mL of PBS, centrifuge at 800 × g and 4°C for 10 min, and discard the supernatant after centrifugation.

[0261] 2.3 Lysis of tissue

[0262] Add 4 mL of cell lysis buffer to the washed tissue pellet and incubate on ice for 8 min.

[0263] 2.4 Screening

[0264] After the lysis is completed, the cell suspension is filtered using a 40 μm cell sieve and transferred to a new 15 mL centrifuge tube. The filtrate is centrifuged at 800 × g and 4° C. for 8 min, and the supernatant is discarded.

[0265] 2.5 Washing

[0266] Add 6 mL of washing buffer to the nuclear pellet, centrifuge at 800 × g and 4°C for 8 min, and discard the supernatant after centrifugation.

[0267] 2.6 Washing

[0268] Add 6 mL of wash buffer to the nuclear pellet from the previous step and centrifuge at 800 × g, 4°C for 5 min. Discard the supernatant. Add 1.5 mL of final buffer to the final nuclear pellet and resuspend it to obtain the nuclear suspension for use in the instrument.

[0269] 2.7 Microscopic examination

[0270] 0.4% trypan blue (Thermo Fisher Scientific, T10282) was used for microscopic examination: 1 μL of trypan blue solution was mixed with 9 μL of cell nucleus suspension and then examined under a microscopic microscope.

[0271] 3.1 Prepare reagents

[0272] Invitrogen-exosomes purification reagent, Exosomal RNA Isolation Kit, PBS

[0273] Prepare single cell nuclei suspension as follows 3.2-3.4

[0274] 3.2 Defragmentation

[0275] Centrifuge the supernatant from the second centrifugation in step 1.5 above at 16,000 × g, 4°C, for 10 min. After centrifugation, collect the supernatant into a new centrifuge tube.

[0276] 3.3 Incubation

[0277] The supernatant after centrifugation in 3.2 was mixed with Invitrogen reagent and incubated at 4°C for 10 h.

[0278] 3.4 Recycling

[0279] The mixture after incubation in 3.3 was centrifuged at 10,000 × g and 4°C for 60 minutes. The precipitate was recovered after centrifugation and resuspended in PBS buffer.

[0280] 3.5 RNA extraction and quantification

[0281] RNA was extracted from the exosomes obtained in step 3.4 using a kit. All procedures were performed according to the kit instructions (Exosomal RNA Isolation Kit). The obtained RNA was quantified using a NanoDrop 2000 to determine the amount of RNA obtained.

[0282] Results and analysis:

[0283] The above experimental results show that the method of the present invention allows the simultaneous preparation of exosomes, single-cell suspensions, and single-nucleus suspensions from the same intrahepatic bile duct tissue. After preparation, the single-cell suspensions and single-nucleus suspensions, after microscopic examination with trypan blue staining, showed that the cell and nucleus concentrations, debris and impurity ratios, and clumping ratios all met the requirements for single-cell sequencing by 10x Genomics. Furthermore, the small RNA isolated from the exosomes and the resulting libraries also met the requirements for NGS sequencing (see Table 1).

[0284] Comparative Example 1

[0285] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0286] 1. Preparation of single cell nucleus suspension

[0287] 1.1 Prepare reagents, instruments, and consumables

[0288] Before sampling, prepare centrifuge tubes, scissors, culture dishes, PBS, a centrifuge, etc. Prepare lysis buffer and wash buffer according to the 10X single-cell nucleus preparation method, and pre-cool the prepared solutions on ice.

[0289] The components of the lysis buffer include (final concentration): 10 mM Tris-HCl (pH = 7.5), 1 mM CaCl2, 5 mM NaCl, 0.1% to 0.2% NP40, 21 mM MgCl2, and 0.4 U / μL RNase Inhibitor.

[0290] The washing buffer contained the following components (final concentrations): 10 mM Tris-HCl (pH=7.5), 1 mM CaCl2, 5 mM NaCl, 21 mM MgCl2, 0.4 U / μL RNase Inhibitor, and 1% to 2% (v / v) BSA.

[0291] 1.2 Sample cleaning

[0292] After the fresh tissue was removed, it was rinsed three times with ice-cold PBS until no obvious blood residue was seen.

[0293] 1.3 Preparation of cell nucleus suspension

[0294] While adding 2 mL of lysis buffer to the culture dish, use sterile scissors to mince the mouse kidney tissue in the culture dish. After mincing, let the tissue rest on ice for 7 minutes. After lysis, add wash buffer and filter through a 40 μm pore size cell sieve. Transfer the filtrate to a new 15 mL centrifuge tube and centrifuge at 1000 × g and 4°C for 10 minutes. Remaining tissue is recovered and used to prepare a single cell suspension. After centrifugation, the nuclear pellet is resuspended in wash buffer and then washed and purified to prepare a single cell nuclear suspension.

[0295] 1.4 Quality inspection of cell nuclear suspension

[0296] Mix 9 μL of the cell nucleus suspension obtained in step 1.3 above with 1 μL of trypan blue staining solution and examine under a microscope to determine the final cell nucleus concentration, fragment ratio, and cell nucleus agglomeration ratio.

[0297] 2. Single cell nucleus suspension

[0298] 2.1 37℃ standstill

[0299] Use a pipette to transfer the prepared single cell nucleus suspension into a 15 mL centrifuge tube and place it at 37°C for 5 minutes.

[0300] 2.2 RNA extraction

[0301] After standing, the single cell nucleus suspension was subjected to nuclear RNA extraction.

[0302] 2.3 RNA Quality Control

[0303] The extracted nuclear RNA was subjected to nanodrop and 4150 quality inspection.

[0304] 2.4 Microscopic examination

[0305] Take 9 μL of the single cell nucleus suspension after standing in the above step and mix it with 1 μL of trypan blue solution, and then perform microscopic examination.

[0306] The results showed that nuclear RNA did not degrade after being placed at 37°C for 5 minutes.

[0307] Comparative Example 2

[0308] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0309] Except that the standing time was changed from 5 min to 10 min, the other operations were exactly the same as those in Comparative Example 1.

[0310] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration did not increase significantly compared with Comparative Example 1, and the nuclear RNA did not degrade after being stored at 37°C for 10 minutes.

[0311] Comparative Example 3

[0312] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0313] Except that the standing time was changed from 5 min to 15 min, the other operations were exactly the same as those in Comparative Example 1.

[0314] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration did not increase significantly compared with Comparative Example 1, and the nuclear RNA did not degrade after being stored at 37°C for 15 minutes.

[0315] Comparative Example 4

[0316] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0317] Except that the standing time was changed from 5 min to 20 min, the other operations were exactly the same as those in Comparative Example 1.

[0318] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration did not increase significantly compared with Comparative Example 1, and the nuclear RNA did not degrade after being stored at 37°C for 20 minutes.

[0319] Comparative Example 5

[0320] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0321] Except that the standing time was changed from 5 min to 25 min, the other operations were exactly the same as those in Comparative Example 1.

[0322] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration were slightly higher than those in Comparative Example 1. After the nuclear RNA was stored at 37° C. for 25 minutes, the RNA was not degraded.

[0323] Comparative Example 6

[0324] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0325] Except that the standing time was changed from 5 min to 30 min, the other operations were exactly the same as those in Comparative Example 1.

[0326] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration increased compared with Comparative Example 1. After the nuclear RNA was stored at 37° C. for 30 minutes, the main peak of RNA showed a slight shift.

[0327] Comparative Example 7

[0328] Intrahepatic bile duct tissue was used as a sample. After the single cell nucleus suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0329] Except that the standing time was changed from 5 min to 35 min, the other operations were exactly the same as those in Comparative Example 1.

[0330] The results showed that as the storage time of the single cell nucleus suspension increased, the ratio of nuclear fragment impurities and the ratio of agglomeration increased compared with Comparative Example 1. After the nuclear RNA was stored at 37° C. for 35 minutes, the RNA began to degrade.

[0331] Comparative Example 8

[0332] Referring to the existing technology provided in the article "Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR", intrahepatic bile duct tissue was used as a sample, and the tissue was broken by homogenization and then exosomes were purified.

[0333] 1. Exosome Preparation

[0334] 1.1 Prepare reagents, instruments, and consumables

[0335] 100 mM ammonium acetate (AA) buffer, bullet blender homogenizer (NextAdvance, NY, USA), 200 mg of 0.9–2.00 mm particles, protease inhibitors, centrifuge tubes, culture dishes, PBS, centrifuge, etc.

[0336] 1.2 Sample cleaning

[0337] After the fresh tissue was removed, it was rinsed three times with ice-cold PBS until no obvious blood residue was seen.

[0338] 1.3 Tissue fragmentation

[0339] Add 500 μL of 100 mM AA buffer and 40 mg of metal beads to the intrahepatic bile duct tissue and homogenize at medium intensity (speed < 6) for 5 minutes. Centrifuge the homogenized sample at 15,000 × g, 4°C, for 10 minutes, and collect the supernatant. Wash the pellet and re-add it to 500 μL of AA buffer for a second round of homogenization for 5 minutes (speed < 8). Centrifuge again at 15,000 × g, 4°C, for 10 minutes, and collect the supernatant.

[0340] 1.4 Exosome isolation and purification

[0341] The supernatant in 1.3 was used to separate and purify exosomes using Qiagen exoEasy Maxi Kit, and all operations were performed according to the instructions.

[0342] 1.5 Exosome Characterization

[0343] The exosomes obtained in step 1.4 were removed and diluted to an appropriate multiple before NTA (nanoparticle tracking analysis) analysis to determine the particle size and concentration. The exosome potential was also determined by zeta potential.

[0344] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0345] 2. Let the exosomes stand at 37°C.

[0346] 2.1 37℃ standstill

[0347] Use a pipette to transfer the prepared exosome suspension into a 1.5 mL centrifuge tube and place it at 37°C for 5 minutes.

[0348] 2.2 Exosome characterization

[0349] After incubation at 37°C for 5 minutes, remove the exosomes obtained in step 2.1 and dilute them to a suitable multiple before performing NTA (nanoparticle tracking analysis) to determine particle size and concentration. Zeta potential was also used to observe changes in the exosomes.

[0350] 2.3 RNA extraction

[0351] After the exosomes were left to stand for 2 h, total RNA was extracted using the Trizol method. All operations were performed according to the reagent instructions.

[0352] 2.4 RNA Quality Control

[0353] The extracted exosomal RNA was subjected to nanodrop and 4150 quality inspection and quantification to determine the total RNA amount obtained.

[0354] The results showed that the total RNA content of exosomes remained unchanged after 5 minutes at 37°C. Furthermore, NTA analysis of the exosomes revealed no significant changes in particle size, concentration, or zeta potential.

[0355] Comparative Example 9

[0356] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0357] Except that the standing time was changed from 5 min to 10 min, the other operations were exactly the same as those in Comparative Example 8.

[0358] The results showed that the total RNA content of exosomes did not change significantly after being stored at 37°C for 10 minutes. Furthermore, NTA analysis of the exosomes showed no significant changes in particle size, concentration, or zeta potential.

[0359] Comparative Example 10

[0360] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0361] Except that the standing time was changed from 5 min to 15 min, the other operations were exactly the same as those in Comparative Example 8.

[0362] The results showed that the total RNA content of exosomes did not change significantly after being stored at 37°C for 15 minutes. Furthermore, NTA analysis of the exosomes showed no significant changes in particle size, concentration, or zeta potential.

[0363] Comparative Example 11

[0364] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0365] Except that the standing time was changed from 5 min to 20 min, the other operations were exactly the same as those in Comparative Example 8.

[0366] Results showed that the total RNA content of exosomes remained unchanged after 20 minutes at 37°C. Furthermore, NTA analysis of the exosomes revealed no significant changes in particle size or concentration. Zeta potential values decreased slightly.

[0367] Comparative Example 12

[0368] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0369] Except that the standing time was changed from 5 min to 25 min, the other operations were exactly the same as those in Comparative Example 8.

[0370] Results showed a slight decrease in total RNA content after exosomes were placed at 37°C for 25 minutes. Furthermore, NTA analysis of the exosomes revealed a slight decrease in particle size and concentration. The zeta potential was also slightly lower than that of Comparative Example 11.

[0371] Comparative Example 13

[0372] Intrahepatic bile duct tissue was used as a sample. After the exosome suspension was prepared, it was placed at 37°C without any enzyme treatment.

[0373] Except that the standing time was changed from 5 min to min, the other operations were exactly the same as those in Comparative Example 8.

[0374] Results showed a slight decrease in total RNA content after exosomes were placed at 37°C for 30 minutes. Furthermore, NTA analysis of the exosomes revealed a slight decrease in particle size and concentration. The zeta potential was also slightly lower than that of Comparative Example 12.

[0375] Comparative Example 14

[0376] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0377] Referring to the existing technology provided in the article "Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR", intrahepatic bile duct tissue was used as a sample, and the tissue was broken by homogenization and then exosomes were purified.

[0378] 1. Exosome Preparation

[0379] 1.2 Prepare reagents, instruments, and consumables

[0380] 100 mM ammonium acetate (AA) buffer, bullet blender homogenizer (NextAdvance, NY, USA), 200 mg of 0.9–2.00 mm particles, protease inhibitors, centrifuge tubes, culture dishes, PBS, centrifuge, etc.

[0381] 1.2 Sample cleaning

[0382] After the fresh tissue was removed, it was rinsed three times with ice-cold PBS until no obvious blood residue was seen.

[0383] 1.3 Tissue fragmentation

[0384] Add 500 μL of 100 mM AA buffer and 40 mg of metal beads to the intrahepatic bile duct tissue and homogenize at medium intensity (speed <6) for 5 minutes. After homogenization, centrifuge the homogenized sample at 15,000 × g, 4°C, for 10 minutes. Collect the supernatant after centrifugation. The remaining tissue is retained for subsequent preparation of single cell and single cell nucleus suspensions. After washing, the particles are placed back in 500 μL of AA buffer and homogenized for a second round for 5 minutes (speed less than 8). Then centrifuge again at 15,000 × g, 4°C, for 10 minutes. Collect the supernatant after centrifugation.

[0385] 1.4 Exosome isolation and purification

[0386] The supernatant in 1.3 was used to separate and purify exosomes using Qiagen exoEasy Maxi Kit, and all operations were performed according to the instructions.

[0387] 1.5 Exosome Characterization

[0388] The exosomes obtained in 1.4 were taken out and diluted to a suitable multiple and then subjected to NTA (nanoparticle tracking analysis) detection to determine the particle size and concentration. At the same time, the exosome potential value was determined by zeta potential.

[0389] 2. Single-cell suspension preparation

[0390] 2.1 Prepare reagents

[0391] Culture medium, collagenase II solution, PBS.

[0392] 2.2 Sample cleaning

[0393] Take the remaining tissue from step 1.3 above, wash it with PBS, and centrifuge it at 1000×g at 4°C for 5 min. Discard the supernatant after centrifugation.

[0394] 2.3 Tissue enzymatic hydrolysis

[0395] Add 4 mL of pre-cooled culture medium to the tissue, add collagenase II (v / v) solution with a final concentration of 0.2%, mix by inversion, and place in a hybridization oven at 37°C for 10 minutes of digestion and enzymatic hydrolysis.

[0396] 2.4 End of enzymatic hydrolysis

[0397] Use a pipette to pipette the tissue, enzyme, and culture medium mixture. Allow the tissue to settle completely. Pipette the supernatant and filter through a 40 μm mesh into a new 15 mL centrifuge tube. Reserve the tissue pellet at the bottom for nuclei preparation. Centrifuge the tube containing the filtrate at 500 × g and 4°C for 10 minutes. Discard the supernatant after centrifugation.

[0398] 2.5 Microscopic examination

[0399] 1 mL of pre-cooled culture medium was added to resuspend the cell pellet, and 0.4% trypan blue (Thermo Fisher Scientific, T10282) was used for microscopic examination: 1 μL of trypan blue solution was mixed with 9 μL of cell suspension, and then microscopic examination was performed after mixing.

[0400] The results showed that after preparation, the single-cell suspension was stained with trypan blue and examined under a microscope. The total cell volume, the proportion of debris and impurities, and the proportion of agglomerates were insufficient, and could not meet the single-cell sequencing requirements of 10x Genomics.

[0401] 3. Preparation of single cell nucleus suspension

[0402] 3.1 Sample cleaning

[0403] Take the remaining tissue from 2.4 above, wash it with PBS, and centrifuge it at 1000×g at 4°C for 5 min. Discard the supernatant after centrifugation.

[0404] 3.2 Preparation of nuclear suspension

[0405] The preparation method is completely consistent with that of Comparative Example 1.

[0406] 3.3 Microscopic examination

[0407] 1 mL of pre-cooled culture medium was added to resuspend the cell nuclear pellet, and 0.4% trypan blue (Thermo Fisher Scientific, T10282) was used for microscopic examination: 1 μL of trypan blue solution was mixed with 9 μL of cell nuclear suspension, and then microscopic examination was performed after mixing.

[0408] The results showed that after the preparation, the single cell nucleus suspension was stained with trypan blue and examined under a microscope. Basically, no cell nuclei could be observed and the total amount of cell nuclei was very small.

[0409] Comparative Example 15

[0410] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0411] Except that the enzymatic hydrolysis time was changed from 10 min to 20 min, the remaining operations were exactly the same as those in Comparative Example 14.

[0412] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0413] Comparative Example 16

[0414] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0415] Except that the enzymatic hydrolysis time was changed from 10 min to 30 min, the remaining operations were exactly the same as those in Comparative Example 14.

[0416] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0417] Comparative Example 17

[0418] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0419] Except for replacing the enzyme with collagenase IV solution, the remaining operations were exactly the same as those in Comparative Example 14.

[0420] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0421] Comparative Example 18

[0422] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0423] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 17.

[0424] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0425] Comparative Example 19

[0426] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0427] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 17.

[0428] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0429] Comparative Example 20

[0430] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0431] Except for replacing the enzyme with collagenase IV + collagenase II solution, the other operations are exactly the same as those in Comparative Example 14.

[0432] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0433] Comparative Example 21

[0434] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0435] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 20.

[0436] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0437] Comparative Example 22

[0438] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by homogenization.

[0439] Except that the enzymatic hydrolysis time was replaced with 30 min, the remaining operations were exactly the same as those in Comparative Example 20.

[0440] The results showed that after preparation, both single cells and single cell nuclei did not meet the single-cell sequencing requirements of 10x Genomics.

[0441] Comparative Example 23

[0442] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0443] Referring to the existing technology provided in the article "Tissue-derived extracellular vesicles: Research progress from isolation to application", intrahepatic bile duct tissue was used as a sample and exosomes were purified using enzymatic hydrolysis.

[0444] 1. Preparation of single-cell suspension

[0445] 1.1 Reagent Preparation

[0446] PBS, culture medium, collagenase D

[0447] 1.2 Sample preparation

[0448] The intrahepatic bile duct tissue was obtained, washed with PBS, wiped dry, and placed in a culture dish. The culture dish was placed on an ice plate, and the tissue was washed with pre-cooled culture medium. After completion, the washing solution was discarded.

[0449] 1.3 Tissue fragmentation

[0450] Use sterile scissors to mince the intrahepatic bile duct tissue. Stop when the tissue is chopped into a paste and transfer the minced tissue pieces in the culture dish to a new 15 mL centrifuge tube.

[0451] 1.4 Tissue enzymatic hydrolysis

[0452] Add 8 mL of pre-cooled culture medium and a collagenase D (v / v) solution with a final concentration of 0.2%. Mix thoroughly by inverting the tube, and place in a hybridization oven at 37°C for 5 minutes for enzymatic digestion.

[0453] 1.5 End of enzymatic hydrolysis

[0454] After standing at room temperature, wait for the tissue blocks to completely settle, and use a pipette to remove the supernatant and transfer it to a new 15mL centrifuge tube. Keep the tissue pellet at the bottom for the preparation of a single cell nucleus suspension. Centrifuge the supernatant for the first time at 3000×g and 4°C for 10 minutes. After the centrifugation is completed, transfer the supernatant to a new centrifuge tube and centrifuge for a second time at 16000×g and 4°C for 10 minutes. The first cell pellet is retained for later use. After the second centrifugation is completed, collect the supernatant from the second centrifugation for subsequent exosome preparation. Resuspend the first and second cell pellets in 4mL of culture medium and transfer them together to a new centrifuge tube for the subsequent preparation of a single cell suspension.

[0455] 1.6 Sieving

[0456] The cell pellet resuspended in 1.5 was pipetted through a 40 μm mesh filter into a new 15 mL centrifuge tube. After sieving, the pellet was centrifuged at 500 × g and 4°C for 10 min. The supernatant was discarded.

[0457] 1.7 Lysis of red blood cells

[0458] Add 8 mL of red blood cell lysis buffer to the cell pellet, mix thoroughly by pipetting, and let stand at room temperature for 5 minutes. After lysis, centrifuge at 500 × g, 4°C, for 5 minutes. Discard the supernatant.

[0459] 2. Preparation of single cell nucleus suspension

[0460] 2.1 Sample cleaning

[0461] Take the remaining tissue from step 1.5 above, wash it with PBS, and centrifuge it at 1000×g at 4°C for 5 min. Discard the supernatant after centrifugation.

[0462] 2.2 Preparation of nuclear suspension

[0463] The preparation method is completely consistent with that of Comparative Example 1.

[0464] 2.3 Microscopic examination

[0465] 1 mL of pre-cooled culture medium was added to resuspend the cell nuclear pellet, and 0.4% trypan blue (Thermo Fisher Scientific, T10282) was used for microscopic examination: 1 μL of trypan blue solution was mixed with 9 μL of cell nuclear suspension, and then microscopic examination was performed after mixing.

[0466] 2.4 Nuclear RNA extraction quality inspection

[0467] Total RNA was extracted from the isolated single cell nuclei using the Trizol method, and all operations were performed according to the reagent instructions.

[0468] 3. Exosome isolation and purification

[0469] 3.1 Exosome isolation

[0470] The supernatant in 1.5 above was used to separate and purify exosomes using Qiagen-exoEasy Maxi Kit.

[0471] 3.2 Exosome characterization

[0472] The exosomes obtained in 3.1 were taken out and diluted to an appropriate multiple before NTA (nanoparticle tracking analysis) analysis to determine the particle size and concentration. The exosome potential was also determined by zeta potential.

[0473] 3.2 Exosome RNA extraction

[0474] Total RNA was extracted from the isolated exosomes using the Trizol method, and all operations were performed according to the reagent instructions.

[0475] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0476] Comparative Example 24

[0477] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0478] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 23.

[0479] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0480] Comparative Example 25

[0481] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0482] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 23.

[0483] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0484] Comparative Example 26

[0485] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0486] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 23.

[0487] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0488] Comparative Example 27

[0489] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0490] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 23.

[0491] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0492] Comparative Example 28

[0493] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0494] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 23.

[0495] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak had shifted slightly. The cell number in the cell suspension was insufficient to meet the requirements of 10x Genomics single-cell sequencing. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0496] Comparative Example 29

[0497] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0498] Except that the enzymatic solution was replaced with collagenase II, the rest of the operations were exactly the same as those in Comparative Example 23.

[0499] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0500] Comparative Example 30

[0501] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0502] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 29.

[0503] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0504] Comparative Example 31

[0505] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0506] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 29.

[0507] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0508] Comparative Example 32

[0509] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0510] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 29.

[0511] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0512] Comparative Example 33

[0513] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0514] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 29.

[0515] The results showed that after preparation, the single cell nuclear suspension was stained with trypan blue and examined under a microscope, which could meet the requirements of 10x Genomics for single cell nuclear sequencing. The number of cells in the single cell suspension was insufficient and could not meet the requirements of 10x Genomics for single cell sequencing. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0516] Comparative Example 34

[0517] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0518] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 29.

[0519] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0520] Comparative Example 35

[0521] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0522] Except that the enzymatic solution was replaced with collagenase IV, the rest of the operations were exactly the same as those in Comparative Example 23.

[0523] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0524] Comparative Example 36

[0525] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0526] Except that the enzymatic hydrolysis time was replaced with 10 min, the remaining operations were exactly the same as those in Comparative Example 35.

[0527] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0528] Comparative Example 37

[0529] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0530] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 35.

[0531] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0532] Comparative Example 38

[0533] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0534] Except that the enzymatic hydrolysis time was replaced with 20 min, the remaining operations were exactly the same as those in Comparative Example 35.

[0535] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0536] Comparative Example 39

[0537] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0538] Except that the enzymatic hydrolysis time was replaced with 25 min, the remaining operations were exactly the same as those in Comparative Example 35.

[0539] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0540] Comparative Example 40

[0541] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0542] Except that the enzymatic hydrolysis time was replaced with 30 min, the remaining operations were exactly the same as those in Comparative Example 35.

[0543] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes was also too low to meet the requirements for small RNA library construction.

[0544] Comparative Example 41

[0545] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0546] Except that the enzymatic hydrolysis solution was replaced with hyaluronidase, the remaining operations were exactly the same as those in Comparative Example 23.

[0547] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0548] Comparative Example 42

[0549] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0550] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 41.

[0551] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0552] Comparative Example 43

[0553] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0554] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 41.

[0555] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0556] Comparative Example 44

[0557] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0558] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 41.

[0559] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0560] Comparative Example 45

[0561] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0562] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 41.

[0563] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0564] Comparative Example 46

[0565] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0566] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 41.

[0567] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0568] Comparative Example 47

[0569] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0570] Except that the enzymatic solution was replaced with trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0571] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0572] Comparative Example 48

[0573] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nucleus suspensions were prepared by enzymatic hydrolysis.

[0574] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 47.

[0575] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0576] Comparative Example 49

[0577] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0578] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 47.

[0579] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0580] Comparative Example 50

[0581] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0582] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 47.

[0583] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0584] Comparative Example 51

[0585] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0586] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 47.

[0587] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0588] Comparative Example 52

[0589] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0590] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 47.

[0591] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0592] Comparative Example 53

[0593] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0594] Except that the enzymatic hydrolysate was replaced with papain, the remaining operations were exactly the same as those in Comparative Example 23.

[0595] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0596] Comparative Example 54

[0597] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0598] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 53.

[0599] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0600] Comparative Example 55

[0601] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0602] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 53.

[0603] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0604] Comparative Example 56

[0605] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0606] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 53.

[0607] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0608] Comparative Example 57

[0609] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0610] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 53.

[0611] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0612] Comparative Example 58

[0613] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0614] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 53.

[0615] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0616] Comparative Example 59

[0617] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0618] Except that the enzymatic solution was replaced with collagenase D and collagenase II, the rest of the operations were exactly the same as those in Comparative Example 23.

[0619] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0620] Comparative Example 60

[0621] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0622] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 59.

[0623] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0624] Comparative Example 61

[0625] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0626] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 59.

[0627] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0628] Comparative Example 62

[0629] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0630] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 59.

[0631] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0632] Comparative Example 63

[0633] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0634] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 59.

[0635] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0636] Comparative Example 64

[0637] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0638] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 59.

[0639] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0640] Comparative Example 65

[0641] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0642] Except that the enzymatic solution was replaced with collagenase D and collagenase IV, the rest of the operations were exactly the same as those in Comparative Example 23.

[0643] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0644] Comparative Example 66

[0645] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0646] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in the comparative example.

[0647] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0648] Comparative Example 67

[0649] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0650] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in the comparative example.

[0651] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0652] Comparative Example 68

[0653] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0654] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in the comparative example.

[0655] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0656] Comparative Example 69

[0657] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0658] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in the comparative example.

[0659] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0660] Comparative Example 70

[0661] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0662] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in the comparative example.

[0663] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0664] Comparative Example 71

[0665] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0666] Except that the enzymatic solution was replaced with collagenase D and hyaluronidase, the other operations were exactly the same as those in Comparative Example 23.

[0667] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0668] Comparative Example 72

[0669] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0670] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in the comparative example.

[0671] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0672] Comparative Example 73

[0673] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0674] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in the comparative example.

[0675] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0676] Comparative Example 74

[0677] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0678] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in the comparative example.

[0679] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0680] Comparative Example 75

[0681] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0682] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in the comparative example.

[0683] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0684] Comparative Example 76

[0685] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0686] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in the comparative example.

[0687] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0688] Comparative Example 77

[0689] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0690] Except that the enzymatic solution was replaced with collagenase D and papain, the remaining operations were exactly the same as those in Comparative Example 23.

[0691] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0692] Comparative Example 78

[0693] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0694] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 77.

[0695] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0696] Comparative Example 79

[0697] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0698] Except for replacing the enzymatic hydrolysis time with 15 min, the remaining operations are exactly the same as those in Comparative Example 77.

[0699] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0700] Comparative Example 80

[0701] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0702] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 77.

[0703] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0704] Comparative Example 81

[0705] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0706] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 77.

[0707] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0708] Comparative Example 82

[0709] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0710] Except for replacing the enzymatic hydrolysis time with 30 min, the remaining operations are exactly the same as those in Comparative Example 77.

[0711] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0712] Comparative Example 83

[0713] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0714] Except that the enzymatic solution was replaced with collagenase D and trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0715] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0716] Comparative Example 84

[0717] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0718] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 83.

[0719] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0720] Comparative Example 85

[0721] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0722] Except for replacing the enzymatic hydrolysis time with 15 min, the remaining operations are exactly the same as those in Comparative Example 83.

[0723] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0724] Comparative Example 86

[0725] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0726] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 83.

[0727] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0728] Comparative Example 87

[0729] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0730] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 83.

[0731] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0732] Comparative Example 88

[0733] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0734] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 83.

[0735] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0736] Comparative Example 89

[0737] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0738] Except for replacing the enzymatic solution with collagenase II and collagenase IV, the rest of the operations are exactly the same as those in Comparative Example 23.

[0739] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0740] Comparative Example 90

[0741] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0742] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 89.

[0743] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0744] Comparative Example 91

[0745] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0746] Except for replacing the enzymatic hydrolysis time with 15 min, the remaining operations are exactly the same as those in Comparative Example 89.

[0747] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0748] Comparative Example 92

[0749] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0750] Except for changing the enzymatic hydrolysis time to 20 min, the remaining operations are exactly the same as those in Comparative Example 89.

[0751] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0752] Comparative Example 93

[0753] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0754] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 89.

[0755] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0756] Comparative Example 94

[0757] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0758] Except for replacing the enzymatic hydrolysis time with 30 min, the remaining operations are exactly the same as those in Comparative Example 89.

[0759] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0760] Comparative Example 95

[0761] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0762] Except that the enzymatic solution was replaced with collagenase II and hyaluronidase, the rest of the operations were exactly the same as those in Comparative Example 23.

[0763] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0764] Comparative Example 96

[0765] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0766] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 95.

[0767] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0768] Comparative Example 97

[0769] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0770] Except for replacing the enzymatic hydrolysis time with 15 min, the remaining operations are exactly the same as those in Comparative Example 95.

[0771] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0772] Comparative Example 98

[0773] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0774] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 95.

[0775] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0776] Comparative Example 99

[0777] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0778] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 95.

[0779] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0780] Comparative Example 100

[0781] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0782] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 95.

[0783] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0784] Comparative Example 101

[0785] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0786] Except that the enzymatic solution was replaced with collagenase II and papain, the rest of the operations were exactly the same as those in Comparative Example 23.

[0787] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0788] Comparative Example 102

[0789] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0790] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 101.

[0791] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0792] Comparative Example 103

[0793] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0794] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 101.

[0795] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0796] Comparative Example 104

[0797] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0798] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 101.

[0799] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0800] Comparative Example 105

[0801] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0802] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 101.

[0803] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0804] Comparative Example 106

[0805] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0806] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 101.

[0807] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0808] Comparative Example 107

[0809] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0810] Except that the enzymatic solution was replaced with collagenase II and trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0811] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0812] Comparative Example 108

[0813] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0814] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 107.

[0815] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0816] Comparative Example 109

[0817] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0818] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 107.

[0819] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0820] Comparative Example 110

[0821] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0822] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 107.

[0823] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0824] Comparative Example 111

[0825] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0826] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 107.

[0827] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0828] Comparative Example 112

[0829] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0830] Except for replacing the enzymatic hydrolysis time with 30 min, the remaining operations are exactly the same as those in Comparative Example 107.

[0831] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0832] Comparative Example 113

[0833] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0834] Except that the enzymatic solution was replaced with collagenase IV and hyaluronidase, the rest of the operations were exactly the same as those in Comparative Example 23.

[0835] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0836] Comparative Example 114

[0837] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0838] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 113.

[0839] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0840] Comparative Example 115

[0841] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0842] Except that the enzymatic hydrolysis time was changed to 15 min, the remaining operations were exactly the same as those in Comparative Example 113.

[0843] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0844] Comparative Example 116

[0845] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0846] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 113.

[0847] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0848] Comparative Example 117

[0849] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0850] Except that the enzymatic hydrolysis time was changed to 25 min, the remaining operations were exactly the same as those in Comparative Example 113.

[0851] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0852] Comparative Example 118

[0853] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0854] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 113.

[0855] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0856] Comparative Example 119

[0857] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0858] Except that the enzymatic solution was replaced with collagenase IV and papain, the remaining operations were exactly the same as those in Comparative Example 23.

[0859] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0860] Comparative Example 120

[0861] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0862] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 119.

[0863] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0864] Comparative Example 121

[0865] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0866] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 119.

[0867] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0868] Comparative Example 122

[0869] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0870] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 119.

[0871] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0872] Comparative Example 123

[0873] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0874] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 119.

[0875] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0876] Comparative Example 124

[0877] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0878] Except for replacing the enzymatic hydrolysis time with 30 min, the remaining operations are exactly the same as those in Comparative Example 119.

[0879] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0880] Comparative Example 125

[0881] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0882] Except that the enzymatic solution was replaced with collagenase IV and trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0883] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0884] Comparative Example 126

[0885] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0886] Except that the enzymatic hydrolysis time was replaced with 10 min, the remaining operations were exactly the same as those in Comparative Example 125.

[0887] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0888] Comparative Example 127

[0889] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0890] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 125.

[0891] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0892] Comparative Example 128

[0893] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0894] Except that the enzymatic hydrolysis time was replaced with 20 min, the remaining operations were exactly the same as those in Comparative Example 125.

[0895] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0896] Comparative Example 129

[0897] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0898] Except that the enzymatic hydrolysis time was replaced with 25 min, the remaining operations were exactly the same as those in Comparative Example 125.

[0899] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0900] Comparative Example 130

[0901] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0902] Except that the enzymatic hydrolysis time was replaced with 30 min, the remaining operations were exactly the same as those in Comparative Example 125.

[0903] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0904] Comparative Example 131

[0905] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0906] Except that the enzymatic hydrolysis solution was replaced with hyaluronidase and papain, the other operations were exactly the same as those in Comparative Example 23.

[0907] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0908] Comparative Example 132

[0909] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0910] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 131.

[0911] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0912] Comparative Example 133

[0913] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0914] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 131.

[0915] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0916] Comparative Example 134

[0917] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0918] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 131.

[0919] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0920] Comparative Example 135

[0921] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0922] Except that the enzymatic hydrolysis time was replaced with 25 min, the remaining operations were exactly the same as those in Comparative Example 131.

[0923] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0924] Comparative Example 136

[0925] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0926] Except that the enzymatic hydrolysis time was changed to 30 min, the remaining operations were exactly the same as those in Comparative Example 131.

[0927] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0928] Comparative Example 137

[0929] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0930] Except that the enzymatic solution was replaced with hyaluronidase and trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0931] The results showed that after preparation, the single cell nuclear suspension was stained with trypan blue and examined under a microscope, which could meet the requirements of 10x Genomics for single cell nuclear sequencing. The number of cells in the single cell suspension was insufficient and could not meet the requirements of 10x Genomics for single cell sequencing. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0932] Comparative Example 138

[0933] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0934] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 137.

[0935] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0936] Comparative Example 139

[0937] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0938] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 137.

[0939] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0940] Comparative Example 140

[0941] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0942] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 137.

[0943] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0944] Comparative Example 141

[0945] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0946] Except for replacing the enzymatic hydrolysis time with 25 min, the remaining operations are exactly the same as those in Comparative Example 137.

[0947] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0948] Comparative Example 142

[0949] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0950] Except that the enzymatic hydrolysis time was replaced with 30 min, the remaining operations were exactly the same as those in Comparative Example 137.

[0951] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0952] Comparative Example 143

[0953] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0954] Except that the enzymatic hydrolysis solution was replaced with papain and trypsin, the remaining operations were exactly the same as those in Comparative Example 23.

[0955] The results showed that after preparation, the single cell nuclear suspension was stained with trypan blue and examined under a microscope, which could meet the requirements of 10x Genomics for single cell nuclear sequencing. The number of cells in the single cell suspension was insufficient and could not meet the requirements of 10x Genomics for single cell sequencing. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0956] Comparative Example 144

[0957] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0958] Except that the enzymatic hydrolysis time was changed to 10 min, the remaining operations were exactly the same as those in Comparative Example 143.

[0959] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0960] Comparative Example 145

[0961] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0962] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 143.

[0963] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0964] Comparative Example 146

[0965] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0966] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 143.

[0967] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0968] Comparative Example 147

[0969] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0970] Except that the enzymatic hydrolysis time was replaced with 25 min, the remaining operations were exactly the same as those in Comparative Example 143.

[0971] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0972] Comparative Example 148

[0973] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0974] Except that the enzymatic hydrolysis time was replaced with 30 min, the remaining operations were exactly the same as those in Comparative Example 143.

[0975] The results showed that after the single-cell nuclear suspension was prepared and subjected to RNA extraction quality inspection, the main RNA peak shifted slightly. The single-cell suspension contained insufficient cells to meet the requirements for single-cell sequencing at 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0976] Comparative Example 149

[0977] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0978] Except that the enzymatic hydrolysis solution was replaced with collagenase D, collagenase II, and collagenase IV, the remaining operations were exactly the same as those in Comparative Example 23.

[0979] The results showed that after preparation, the single cell nuclear suspension was stained with trypan blue and examined under a microscope, which could meet the requirements of 10x Genomics for single cell nuclear sequencing. The number of cells in the single cell suspension was insufficient and could not meet the requirements of 10x Genomics for single cell sequencing. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0980] Comparative Example 150

[0981] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0982] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 149.

[0983] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0984] Comparative Example 151

[0985] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0986] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 149.

[0987] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[0988] Comparative Example 152

[0989] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0990] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 149.

[0991] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[0992] Comparative Example 153

[0993] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0994] Except that the enzymatic solution was replaced with collagenase D, collagenase II, and hyaluronidase, the remaining operations were exactly the same as those in Comparative Example 23.

[0995] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[0996] Comparative Example 154

[0997] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[0998] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 153.

[0999] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1000] Comparative Example 155

[1001] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1002] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 153.

[1003] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1004] Comparative Example 156

[1005] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1006] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 153.

[1007] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1008] Comparative Example 157

[1009] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1010] Except that the enzymatic solution was replaced with collagenase D, collagenase II and papain, the other operations were exactly the same as those in Comparative Example 23.

[1011] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1012] Comparative Example 158

[1013] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1014] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 157.

[1015] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1016] Comparative Example 159

[1017] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1018] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 157.

[1019] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1020] Comparative Example 160

[1021] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1022] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 157.

[1023] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1024] Comparative Example 161

[1025] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1026] Except that the enzymatic solution was replaced with collagenase D, collagenase II and trypsin, the other operations were exactly the same as those in Comparative Example 23.

[1027] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1028] Comparative Example 162

[1029] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1030] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 161.

[1031] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1032] Comparative Example 163

[1033] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1034] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 161.

[1035] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1036] Comparative Example 164

[1037] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1038] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 161.

[1039] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1040] Comparative Example 165

[1041] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1042] Except that the enzymatic solution was replaced with collagenase II, collagenase IV and hyaluronidase, the rest of the operations were exactly the same as those in Comparative Example 23.

[1043] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1044] Comparative Example 166

[1045] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1046] Except that the enzymatic hydrolysis time was replaced with 10 min, the remaining operations were exactly the same as those in Comparative Example 165.

[1047] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1048] Comparative Example 167

[1049] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1050] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 165.

[1051] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The total amount of exosomes met the requirements for small RNA library construction.

[1052] Comparative Example 168

[1053] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1054] Except that the enzymatic hydrolysis time was changed to 20 min, the remaining operations were exactly the same as those in Comparative Example 165.

[1055] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1056] Comparative Example 169

[1057] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1058] Except that the enzymatic solution was replaced with collagenase II, collagenase IV and papain, the other operations were exactly the same as those in Comparative Example 23.

[1059] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1060] Comparative Example 170

[1061] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1062] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 169.

[1063] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1064] Comparative Example 171

[1065] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1066] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 169.

[1067] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1068] Comparative Example 172

[1069] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1070] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 169.

[1071] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1072] Comparative Example 173

[1073] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1074] Except that the enzymatic solution was replaced with collagenase II, collagenase IV and pancreatic leukemia enzyme, the other operations were exactly the same as those in Comparative Example 23.

[1075] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1076] Comparative Example 174

[1077] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1078] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 173.

[1079] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1080] Comparative Example 175

[1081] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1082] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 173.

[1083] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1084] Comparative Example 176

[1085] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1086] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 173.

[1087] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1088] Comparative Example 177

[1089] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1090] Except that the enzymatic solution was replaced with collagenase IV, hyaluronidase and papain, the other operations were exactly the same as those in Comparative Example 23.

[1091] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1092] Comparative Example 178

[1093] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1094] Except for replacing the enzymatic hydrolysis time with 10 minutes, the remaining operations are exactly the same as those in Comparative Example 177.

[1095] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1096] Comparative Example 179

[1097] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1098] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 177.

[1099] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1100] Comparative Example 180

[1101] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1102] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 177.

[1103] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1104] Comparative Example 181

[1105] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1106] Except that the enzymatic solution was replaced with collagenase IV, hyaluronidase and trypsin, the other operations were exactly the same as those in Comparative Example 23.

[1107] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1108] Comparative Example 182

[1109] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1110] Except for replacing the enzymatic hydrolysis time with 10 min, the remaining operations are exactly the same as those in Comparative Example 181.

[1111] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1112] Comparative Example 183

[1113] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1114] Except for replacing the enzymatic hydrolysis time with 15 minutes, the remaining operations are exactly the same as those in Comparative Example 181.

[1115] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1116] Comparative Example 184

[1117] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1118] Except for replacing the enzymatic hydrolysis time with 20 min, the remaining operations are exactly the same as those in Comparative Example 181.

[1119] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1120] Comparative Example 185

[1121] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1122] Except that the enzymatic solution was replaced with hyaluronidase, papain and trypsin, the other operations were exactly the same as those in Comparative Example 23.

[1123] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1124] Comparative Example 186

[1125] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1126] Except that the enzymatic hydrolysis time was replaced with 10 min, the remaining operations were exactly the same as those in Comparative Example 185.

[1127] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. The total amount of exosomes did not meet the requirements for small RNA library construction.

[1128] Comparative Example 187

[1129] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1130] Except that the enzymatic hydrolysis time was replaced with 15 min, the remaining operations were exactly the same as those in Comparative Example 185.

[1131] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. However, the single-cell suspension did not contain enough cells to meet the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes met the requirements for small RNA library construction.

[1132] Comparative Example 188

[1133] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1134] Except that the enzymatic hydrolysis time was replaced with 20 min, the remaining operations were exactly the same as those in Comparative Example 185.

[1135] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The cell count in the single-cell suspension also met the requirements for single-cell sequencing by 10x Genomics. However, the total amount of exosomes did not meet the requirements for small RNA library construction.

[1136] Comparative Example 189

[1137] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1138] Except for adding 0.2% trypsin after enzymatic hydrolysis at 37°C and letting it stand at room temperature for 2 minutes, the other operations are exactly the same as those in Comparative Example 167.

[1139] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The total amount of exosomes met the requirements for small RNA library construction.

[1140] Comparative Example 190

[1141] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1142] Except for adding 0.2% trypsin after enzymatic hydrolysis at 37°C and letting it stand at room temperature for 4 minutes, the other operations are exactly the same as those in Comparative Example 167.

[1143] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The total amount of exosomes met the requirements for small RNA library construction.

[1144] Comparative Example 191

[1145] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1146] Except for adding 0.2% trypsin after enzymatic hydrolysis at 37°C and letting it stand at room temperature for 6 minutes, the other operations are exactly the same as those in Comparative Example 167.

[1147] The results showed that after preparation, the single-cell nuclear suspension, after trypan blue staining and microscopic examination, met the requirements for single-cell nuclear sequencing by 10x Genomics. The total amount of exosomes met the requirements for small RNA library construction.

[1148] Comparative Example 192

[1149] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1150] 1. Preparation of single cell suspension

[1151] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1152] 2. Preparation of single cell nucleus suspension

[1153] The tissue pellet after enzymatic hydrolysis was washed 0 times and then single cell nuclei were prepared.

[1154] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1155] Comparative Example 193

[1156] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1157] 1. Preparation of single cell suspension

[1158] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1159] 2. Preparation of single cell nucleus suspension

[1160] The tissue pellet after enzymatic hydrolysis was washed once and then single cell nuclei were prepared.

[1161] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1162] Comparative Example 194

[1163] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1164] 1. Preparation of single cell suspension

[1165] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1166] 2. Preparation of single cell nucleus suspension

[1167] The tissue pellet after enzymatic hydrolysis was washed twice and then single cell nuclei were prepared.

[1168] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1169] Comparative Example 195

[1170] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1171] 1. Preparation of single cell suspension

[1172] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1173] 2. Preparation of single cell nucleus suspension

[1174] The tissue pellet after enzymatic hydrolysis was washed three times and then single cell nuclei were prepared.

[1175] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1176] Comparative Example 196

[1177] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1178] 1. Preparation of single cell suspension

[1179] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1180] 2. Preparation of single cell nucleus suspension

[1181] The tissue pellet after enzymatic hydrolysis was washed 4 times and then single cell nuclei were prepared.

[1182] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1183] Comparative Example 197

[1184] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1185] 1. Preparation of single cell suspension

[1186] The single cell suspension preparation operation is exactly the same as that of Comparative Example 190.

[1187] 2. Preparation of single cell nucleus suspension

[1188] The preparation procedure of the single cell nucleus suspension is exactly the same as that of Comparative Example 1, except that the main component of the cell nucleus lysis solution is changed from 0.1% to 0.2% NP40 to 0.1% to 0.2% TritonX-100.

[1189] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1190] Comparative Example 198

[1191] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.1% to 0.2% Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1192] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1193] Comparative Example 199

[1194] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.1% to 0.2% NP40+TritonX-100, the other operations are exactly the same as those in Comparative Example 197.

[1195] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1196] Comparative Example 200

[1197] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.1% to 0.2% NP40 + Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1198] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1199] Comparative Example 201

[1200] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.1% to 0.2% TritonX-100+Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1201] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1202] Comparative Example 202

[1203] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.3% NP40 + Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1204] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1205] Comparative Example 203

[1206] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.4% NP40 + Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1207] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1208] Comparative Example 204

[1209] Except for changing the main component of the cell nuclear lysis solution from 0.1% to 0.2% NP40 to 0.5% NP40 + Tween 20, the other operations are exactly the same as those in Comparative Example 197.

[1210] The results showed that the cell nucleus could not meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1211] Comparative Example 205

[1212] Except for changing the Tris-HCl component of the cell nuclear lysis solution and washing buffer solution from 10 mM to 50 mM, the other operations are exactly the same as those in Comparative Example 203.

[1213] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1214] Comparative Example 206

[1215] Except for changing the Tris-HCl component of the cell nuclear lysis solution and the washing buffer solution from 10 mM to 100 mM, the other operations are exactly the same as those in Comparative Example 203.

[1216] The results showed that the cell nucleus could meet the single-cell nucleus sequencing requirements of 10x Genomics.

[1217] Comparative Example 207

[1218] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1219] Exosomes were separated and purified using ultracentrifugation, and the remaining operations were exactly the same as those in Comparative Example 190.

[1220] Comparative Example 208

[1221] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1222] Exosomal RNA Isolation Kit was used to separate and purify exosomes, and the remaining operations were exactly the same as those in Comparative Example 190.

[1223] Comparative Example 209

[1224] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1225] Exosomes were isolated and purified using Norgen-Cell Culture Media Exosome Purification Mini Kit (60500), and the remaining operations were exactly the same as those in Comparative Example 190.

[1226] Comparative Example 210

[1227] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1228] Exosomes were separated and purified using the Enze Kangtai-Exosome Purification Kit (Echo9101A-10ml), and the remaining operations were exactly the same as those in Comparative Example 190.

[1229] Comparative Example 211

[1230] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1231] Exosomes were isolated and purified using Vazyme's VEX Exosome Isolation Reagent, and the remaining operations were exactly the same as those in Comparative Example 190.

[1232] Comparative Example 212

[1233] Intrahepatic bile ducts were used as samples, and exosomes, single-cell suspensions, and single-cell nuclei suspensions were prepared by enzymatic hydrolysis.

[1234] Exosomes were separated and purified using the exosome purification kit (magnetic bead type) from omiget, and the remaining operations were exactly the same as those in comparative example 190.

[1235] Comparative Example 213

[1236] Mouse heart was used as a sample. Except for the difference in tissue samples, all other operations were exactly the same as those in Example 1 of the present invention.

[1237] The results showed that the technology described in this invention was used to simultaneously prepare exosomes, single cell suspensions, and nuclear suspensions from mouse heart tissue. The results also showed that mouse heart tissue could not be sequenced simultaneously for exosome small RNA, single cell sequencing, and single cell nuclear sequencing.

[1238] Comparative Example 214

[1239] Mouse spleen was used as a sample. Except for the difference in tissue samples, all other operations were exactly the same as those in Example 1 of the present invention.

[1240] The results showed that the technology described in this invention was used to simultaneously prepare exosomes, single-cell suspensions, and nuclear suspensions from mouse spleen tissue. The results also showed that simultaneous exosome small RNA sequencing, single-cell sequencing, and single-cell nuclear sequencing could not be performed on mouse heart tissue.

[1241] Furthermore, according to the experimental results of Examples 1 and 2 of the present invention provided in Table 1 of the present invention, it is shown that the experimental method provided by the present invention can simultaneously prepare exosomes, single cell suspensions and single cell nuclei suspensions in human intrahepatic bile duct tissue, and can prepare 300ng of total exosome RNA, 98W cells, and 220W cell nuclei. The results obtained can meet the requirements of various quality indicators of small RNA sequencing, 10x Genomics single cell and single cell nucleus sequencing experiments, and successfully complete the exosome + single cell + single cell nucleus sequencing experiment of the same intrahepatic bile duct tissue. (See Figures 1 to 7 ).

[1242] Table 1 Experimental results of the method of the present invention for simultaneous preparation of exosomes, single cells and cell nuclei suspensions based on the same intrahepatic bile duct tissue

[1243]

[1244]

[1245] The present invention proposes a method for simultaneously preparing exosomes, single-cell suspensions, and single-cell nucleus suspensions in the same human intrahepatic bile duct tissue, and simultaneously performing small RNA sequencing and single-cell sequencing / single-cell nucleus sequencing. A series of comparative experiments, as shown in Comparative Examples 1 to 214, demonstrate that the present invention has outstanding substantive features and significant progress.

[1246] The present invention creatively combines exosome small RNA sequencing with single-cell and single-nucleus sequencing. Because the detergents in the nucleus lysis buffer used to prepare single-cell nucleus suspensions disrupt the phospholipid bilayer, dissolve the cytoplasm and cell membrane, and cause significant cell damage, this can result in insufficient collection of intact cells during subsequent single-cell suspension preparation, failing to meet the 10x Genomics machine requirements. It can also damage extracellular vesicles, making it impossible to meet smallRNA sequencing requirements. To this end, the present invention employs a sequence of preparing single-cell suspensions and exosomes first, followed by single-cell nucleus suspensions, to simultaneously obtain exosomes, single-cell suspensions, and single-nucleus suspensions from the same tissue.

[1247] Comparative Examples 1 to 7 prepared single cell nuclear suspensions according to the 10x Genomics method for preparing cell nuclei, and then the prepared single cell nuclear suspensions were respectively divided into 7 15mL centrifuge tubes and placed at 37°C to simulate the effect of temperature on the cell nuclear suspension during enzymatic hydrolysis. Every 5 minutes, a single cell nuclear suspension was extracted, and the cell nuclear suspension was subjected to trypan blue staining microscopy and RNA extraction quality control. The results showed that as time increased (5min, 10min, 15min, 20min, 25min, 30min, 35min), the ratio of nuclear fragment impurities and the ratio of agglomeration of single cell nuclear suspensions increased; and after 30 minutes, the main peak of RNA shifted slightly, and after 35 minutes, nuclear RNA was degraded. In summary, it is speculated that when performing exo+scRNA-seq&snRNA-seq in the intrahepatic bile duct tissue of the same person, the 37°C enzymatic hydrolysis time must be controlled within 35 minutes when preparing single-cell suspension, and try not to exceed 30 minutes to avoid affecting the quality of subsequent single-cell nuclear suspension.

[1248] Table 2 Changes in the storage time of prepared single cell nucleus suspension at 37°C

[1249]

[1250]

[1251] According to the results of Comparative Examples 1 to 7, to ensure the integrity of high-quality single-cell nuclear RNA, the enzymatic hydrolysis time should be controlled within 30 minutes. Next, the changes in exosomes placed in a 37°C environment were simulated to ensure the quality of the vesicles. As shown in Comparative Examples 8 to 13, the total amount of exosome RNA, exosome NTA results, exosome TEM results, and exosome zeta potential results were used as the main quality control indicators for comprehensive comparison and judgment. Comparative Examples 8 to 13 prepared human intrahepatic bile duct tissue exosomes using the QIAGEN kit according to the tissue exosome isolation method provided in the literature. The prepared exosomes were then divided into 6 1.5mL centrifuge tubes and placed at 37°C to simulate the effect of temperature on extracellular vesicles during the enzymatic hydrolysis process. Every 5 minutes, a portion of exosomes was extracted and the exosomes were subjected to RNA extraction quality control and NTA / zeta potential testing. The results showed that over time (5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes), the total amount of exosomal RNA began to decrease slightly after 25 minutes. Furthermore, after 20 minutes, exosome NTA and zeta potential assays revealed a slight decrease in exosome concentration and a slight change in zeta potential. In summary, it is speculated that if exosomes are prepared using enzymatic methods at 37°C for exosomes, the hydrolysis time must be controlled within 25 minutes and preferably not exceed 20 minutes to avoid affecting the quality of subsequent extracellular vesicles.

[1252] Table 3 Changes in prepared exosomes after storage at 37°C

[1253]

[1254] According to the exosome small RNA sequencing and single cell / single cell nucleus sequencing processes, it is first necessary to try to combine the three preparation methods to achieve the simultaneous preparation of exosomes, single cell suspensions, and single cell nucleus suspensions in the same tissue, and all of which can meet the requirements for sequencing on the machine. According to the existing technology provided in the article "Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR", intrahepatic bile duct tissue is used as a sample, and the tissue is broken by homogenization and then exosomes are purified. Therefore, we first try to prepare exosomes, single cell suspensions, and single cell nucleus suspensions simultaneously by homogenization, as shown in Comparative Examples 14 to 22, using cell viability, total cell / nucleus volume, cell / nucleus fragment ratio, cell / nucleus cluster ratio, and total exosome RNA quantity as quality control indicators. The results of Comparative Example 14 show that the amount of total exosome RNA prepared can meet the requirements for small RNA sequencing, but the quality of the cell / nucleus suspension cannot meet the requirements for 10x Genomics on the machine. Next, the enzymatic hydrolysis formula was tested, as shown in Comparative Examples 14 to 22. Comparative Examples 14 to 16 used 0.2% collagenase II to dissociate the intrahepatic bile duct tissue, and verified the enzymatic hydrolysis time (10min, 20min, 30min) respectively. Comparative Examples 17 to 19 used 0.2% collagenase IV to dissociate the intrahepatic bile duct tissue, and verified the enzymatic hydrolysis time (10min, 20min, 30min) respectively. Comparative Examples 20 to 22 used 0.2% collagenase II + 0.2% collagenase IV enzyme to dissociate the intrahepatic bile duct tissue, and verified the enzymatic hydrolysis time (10min, 20min, 30min) respectively. The results of Comparative Examples 14 to 22 all showed that although the homogenization method can be used to prepare exosomes that meet the requirements of smallRNA sequencing, it is impossible to prepare high-quality single-cell / single-cell nucleus suspensions. The number of cells / nuclei in the single-cell / single-cell nucleus suspensions is insufficient, and the fragmentation rate is high and there are more impurities. It is speculated that the homogenization method may cause certain damage to cells and cell nuclei, so it is impossible to simultaneously prepare single-cell / single-cell nucleus suspensions that meet the requirements of the machine.

[1255] Table 4 Preparation of exosome, single cell and single cell nucleus suspensions using homogenization method

[1256]

[1257]

[1258] Next, we attempted to prepare exosome, single cell, and single nucleus suspensions using enzymatic hydrolysis. As shown in Comparative Examples 23-191, we determined the enzymatic hydrolysis formulas and hydrolysis times necessary to simultaneously prepare all three suspensions. Using 10x Genomics standards, we used the total number of cells / nuclei, the ratio of cell / nuclear fragments, the ratio of cell / nuclear clusters, the quality of nuclear RNA, and the total exosome RNA content as key quality control indicators for comprehensive comparison and assessment. Comparative Examples 23 to 52 used a single enzyme formula (0.5% collagenase D, 0.5% collagenase II, 0.3% collagenase IV, 0.1% hyaluronidase, 0.2% papain, 0.2% trypsin) for dissociation (5 min, 10 min, 15 min, 20 min, 25 min, 30 min). The prepared cell nucleus suspension can meet the 10x Genomics company's machine standard. However, when the enzymatic hydrolysis exceeds 25 min, the nuclear RNA quality control found that the main peak of the nuclear RNA was offset. Based on the results of Comparative Examples 1 to 7, it is speculated that this may be because the 37°C digestion and enzymatic hydrolysis time is too long, which has a certain damage to the nuclear membrane. At the same time, Comparative Examples 23 to 52 were unable to obtain a good single cell suspension (cell viability and cell amount could not meet the 10x Genomics company's machine standard) and exosomes (the total amount of exosome RNA was insufficient to meet the requirements of small RNA library construction). It is speculated that this may be due to the short dissociation time and insufficient dissociation of a single enzyme. However, prolonged dissociation times resulted in the prepared nuclei suspension failing to meet 10x Genomics' standards for processing. Since no single enzyme formulation could achieve effective dissociation within 25 minutes, yielding high-quality single-cell suspensions and exosomes, we next experimented with two combined enzyme formulations, hoping to achieve the most complete dissociation possible by combining different enzyme cleavage sites, thus avoiding the drawbacks of inadequate dissociation caused by a single enzyme with too short a time.Comparative Examples 59 to 148 used two enzyme formulas (0.5% collagenase D + 0.5% collagenase II, 0.5% collagenase D + 0.3% collagenase IV, 0.5% collagenase D + 0.1% hyaluronidase, 0.5% collagenase D + 0.2% papain, 0.5% collagenase D + 0.2% trypsin, 0.5% collagenase II + 0.3% collagenase IV, 0.5% collagenase II + 0.1% hyaluronidase, 0.5% collagenase II + 0.2% papain, 0.5% collagenase II + 0.2% trypsin). Trypsin, 0.3% collagenase IV + 0.1% hyaluronidase, 0.3% collagenase IV + 0.2% papain, 0.3% collagenase IV + 0.2% trypsin, 0.1% hyaluronidase + 0.2% papain, 0.1% hyaluronidase + 0.2% trypsin, 0.2% papain + 0.2% trypsin) were used for dissociation (5 min, 10 min, 15 min, 20 min, 25 min, 30 min). The results showed that the prepared cell nucleus suspension could meet the 10x Genomics machine standard. However, when the enzymatic hydrolysis exceeded 20 min, the nuclear RNA quality control found that the main peak of the nuclear RNA shifted. Comprehensive comparison of the results of Examples 1 to 7 speculated that this may be due to the damage to the nuclear membrane caused by the prolonged enzymatic hydrolysis at 37 ° C and the damage of the complex enzyme itself to the nuclear membrane. At the same time, the two enzyme formulas could not achieve effective single cell suspension and exosome preparation within 30 min. Therefore, we then tried a combination of three enzymes, hoping to achieve sufficient dissociation within 20 minutes by combining three different enzymes, so as to avoid the defect of insufficient dissociation caused by a single / two enzymes with too short a time for enzymatic hydrolysis. Comparative Examples 149 to 187 used three enzyme formulas (0.5% collagenase D + 0.5% collagenase II + 0.3% collagenase IV, 0.5% collagenase D + 0.5% collagenase II + 0.1% hyaluronidase, 0.5% collagenase D + 0.5% collagenase II + 0.2% papain, 0.5% collagenase D + 0.5% collagenase II + 0.1% hyaluronidase, 0.5% collagenase D + 0.5% collagenase II + 0.2% trypsin, 0.5% collagenase II + 0.1% hyaluronidase + 0.2% papain, 0.5% collagenase II + 0.1% hyaluronidase + 0.2% trypsin, 0.1% hyaluronidase + 0.2% papain + 0.2% trypsin) for dissociation (5 min, 10 min, 15 min, 20 min). The results showed that Comparative Example 167 was able to simultaneously prepare exosomes, single-cell suspensions, and single-cell nuclei suspensions from the same intrahepatic bile duct tissue. The remaining comparative examples were unable to achieve the same 20-minute preparation of exosomes, single-cell, and single-cell nuclei suspensions from the same intrahepatic bile duct tissue. However, the single-cell suspensions prepared exhibited a high clumping rate, leading to the subsequent attempt to improve the quality of the single-cell suspension by adding pancreatic enzymes.Comparative Examples 189 to 191 added 0.2% trypsin after enzymatic hydrolysis and stood at room temperature for 2 minutes, 4 minutes, and 6 minutes, respectively. The results of Comparative Examples 189 to 191 showed that the agglomeration rate was reduced after the addition of trypsin, presumably because trypsin can destroy the intercellular connection, thereby reducing the agglomeration rate. However, when the enzymatic hydrolysis time exceeded 4 minutes, the cell viability was reduced. According to the 10xGenomics company standards, the total cell volume, fragment ratio, agglomeration ratio, total cell nucleus volume, cell nucleus fragment ratio, cell nucleus agglomeration rate, cell nucleus RNA quality, and total exosome RNA content were used as the main quality control indicators to determine the formula for preparing single cell suspension. Therefore, the method for preparing single cell suspension was finally determined, that is, after the intrahepatic bile duct tissue was minced with sterile scissors, 8 mL of pre-cooled culture medium was added, and a final concentration of 0.5% collagenase II (v / v) solution, 0.3% collagenase IV solution, and 0.1% hyaluronidase (v / v) solution was added. After mixing by inversion, place in a hybridization oven at 37°C for 15 minutes for digestion and enzymatic hydrolysis. After digestion, add 0.2% (v / v) trypsin solution to the centrifuge tube, mix by inversion, and let it stand at room temperature for 4 minutes.

[1259] Table 5 Preparation of exosomes, single cells and single cell nuclei suspensions using enzymatic hydrolysis

[1260]

[1261]

[1262]

[1263]

[1264]

[1265]

[1266]

[1267]

[1268]

[1269]

[1270]

[1271]

[1272]

[1273]

[1274] When the enzymatic hydrolysis method can simultaneously prepare exosomes, single cell suspensions, and single cell nucleus suspensions, and the enzymatic hydrolysis formula is determined, the preparation and purification of the cell nucleus suspension needs to be explored and optimized. Comparative Examples 192 to 196 compared the pretreatment of the remaining tissue precipitates after the preparation of single cell suspensions and exosomes. The results showed that when the remaining tissue after enzymatic hydrolysis was directly used for single cell nucleus preparation without washing, basically no impurity fragments were observed, and the impurity fragment rate was high, which may be due to the residual enzymes after enzymatic hydrolysis adhering to the remaining tissue and causing damage to the nuclear membrane. When the remaining tissue after enzymatic hydrolysis was washed three times, the amount of cell nuclei prepared was the highest and the fragmentation rate was the lowest. There was no significant difference between washing 4 times and washing 3 times, and a longer washing time increased the risk of cell nucleus fragmentation, so it was finally determined that the number of times the remaining tissue after enzymatic hydrolysis was washed was 3 times.

[1275] Table 6 Comparison of washing times of residual tissue after enzymatic hydrolysis

[1276]

[1277]

[1278] Comparative Examples 197-206 compared methods for preparing cell nuclei. Since microscopic examination of the cell nuclei suspension showed a slightly high fragmentation rate after using the 10X method to prepare cell nuclei from the remaining tissue, it is speculated that this may be caused by the enzymatic hydrolysis method, which slightly affects the integrity of the cell nuclei. Therefore, Comparative Examples 197-206 verified the components of the cell nuclei lysis solution and wash buffer to see if they could improve the quality of the cell nuclei suspension. First, the most important component of the cell nuclei lysis solution is a non-ionic detergent. Therefore, in Comparative Examples 197-198, the main component of the 10X single cell nuclei lysis solution, 0.1%-0.2% NP40, was replaced with 0.1%-0.2% TritonX-100 and 0.1%-0.2% Tween 20, respectively. The results were quality controlled based on the total number of cell nuclei, cell nuclei concentration, fragment impurity ratio, and clumping ratio, as well as whether they met the single-cell sequencing requirements of 10x Genomics. The results showed that the fragmentation rate of the cell nucleus suspension was still high and had not improved. It is speculated that a single detergent component may be difficult to improve the quality of the single cell nucleus suspension. Some of the fragments in the cell nucleus suspension are plasma membrane fragments generated after the cell membrane is destroyed. A stronger detergent component may be needed to destroy the cell membrane fragments and purify the single cell nucleus suspension. Therefore, a composite detergent component was tried next. Comparative Examples 199-201 respectively replaced the main component of the 10x single cell nucleus lysate, 0.1%-0.2% NP40, with 0.1%-0.2% TritonX-100 + 0.1%-0.2% NP40, 0.1%-0.2% Tween 20 + 0.1%-0.2% NP40, and 0.1%-0.2% TritonX-100 + 0.1%-0.2% Tween 20. The results showed that the quality control results of Comparative Example 200 were higher than those of Comparative Examples 199 and 201, so it was finally determined that the lysate used the combination of detergents TritonX-100 + Tween 20 as the main component. Based on the determination of the formula of the lysate for preparing the cell nuclei, the concentrations of the main components of the cell nuclei lysate were verified in Comparative Examples 202 to 205. The results showed that as the concentration of the detergent increased, the fragmentation rate of the single cell nucleus suspension was reduced compared to Comparative Example 200. When the concentration used was 0.3% to 0.4% NP40 + 0.3% to 0.4% Tween 20, the quality deviation of the single cell nucleus suspension was not large and was basically the same. When the concentration used in Comparative Example 205 was 0.5% NP400 + 0.5% Tween 20, the fragmentation rate of the single cell nucleus suspension began to increase compared to before. It is speculated that this may be because the detergent concentration was too high, and the cell nuclei were excessively lysed and fragmented. Therefore, the concentration of the lysis buffer detergent was finally determined to be 0.3% to 0.4% NP40 + 0.3% to 0.4% Tween 20.At this point, although the quality of the cell nucleus suspension is improved compared with the 10X method, the fragmentation rate is still slightly high. It is speculated that this may be due to the buffer environment in the cell nucleus lysate and the washing buffer. The Tris-HCl concentration is too low, and the cell contents released after the cell membrane rupture cannot be effectively buffered, resulting in changes in the pH of the buffer, triggering denaturation of the surface proteins of the cell nuclear membrane, and ultimately destroying the cell nucleus structure. So next try to adjust the Tris-HCl concentration. Comparative Examples 205-206 replaced the 10mM Tris-HCl in the lysate and washing buffer with 50mM Tris-HCl and 100mM Tris-HCl, respectively. The results show that the fragmentation rate of Comparative Example 205 is lower, and the fragmentation rate of Comparative Example 206 is higher. It is speculated that this may be because the excessively high concentration of Tris-HCl may cause the solution osmotic pressure to change and the cell nucleus to rupture. So it was finally determined that the Tris-HCl concentration was 50mM.

[1279] Table 7 Comparison of methods for preparing single cell nucleus suspension

[1280]

[1281]

[1282]

[1283] After establishing methods for preparing single-cell suspensions and single-cell nuclei suspensions from intrahepatic bile duct tissue, the next step was to improve methods for isolating and purifying exosomes from intrahepatic bile duct tissue. Since there are currently no methods specifically designed for isolating tissue exosomes, we next compared different methods for isolating exosomes from intrahepatic bile duct tissue, using exosome TEM and NTA assay results, total exosome RNA content, and "contamination" (260 / 280) of impurities such as protein in total RNA as quality control indicators, as shown in Comparative Examples 207-212. Methods included ultracentrifugation, magnetic bead methods, and column-based membrane affinity methods. Results showed that the exosome concentrations obtained using the Invitrogen kit, QIAGEN kit, and ultracentrifugation methods were similar. However, considering data on total exosome RNA content and protein contamination, the Invitrogen kit performed best. In summary, after a series of comparisons, the Invitrogen kit was ultimately selected as the method for isolating and purifying exosomes from intrahepatic bile duct tissue.

[1284] Table 8 Comparison of methods for isolating and purifying exosomes

[1285]

[1286]

[1287] Based on the technical solutions for simultaneously preparing exosomes, single-cell suspensions, and single-cell nuclei suspensions in human intrahepatic bile duct tissue established in Comparative Examples 1-212, Comparative Examples 213-214 repeated the same technical solutions on several other types of mouse tissue. The results showed that it was impossible to simultaneously prepare exosomes, single-cell suspensions, and single-cell nuclei suspensions in mouse tissue.

[1288] Table 9 Comparison of exosomes, single cell suspensions and single cell nuclei prepared from different tissues

[1289]

[1290] In summary, the present invention proposes a method for simultaneously preparing exosomes, single cell suspensions, and single cell nucleus suspensions from the same intrahepatic bile duct tissue sample, performing small RNA sequencing, and single cell sequencing & single cell nucleus sequencing. The single cell suspension preparation method (enzymatic hydrolysis formula, enzymatic hydrolysis time), the single cell nucleus suspension preparation method (number of washes of the remaining tissue after enzymatic hydrolysis, lysate, wash buffer formula, lysate concentration, etc.), and the exosomes (purification and separation method) are one-to-one corresponding. Using the methods of Examples 1 and 2 of the present invention, high-quality (i.e., high total amount, low fragmentation rate and impurities, total cell amount 98W, cell suspension fragmentation rate 4%, clumping rate 4%, total cell nuclei 220W, cell nucleus suspension fragmentation rate 5%, clumping rate 5%) single cell & single cell nucleus suspensions and high-quality pure exosomes can be effectively prepared, and the obtained single cell suspension and single cell nucleus suspension can meet the requirements of single cell sequencing experiments, and the obtained exosomes can meet the requirements of small RNA sequencing.

[1291] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A method for simultaneously preparing exosomes, single cell suspension, and single cell nucleus suspension in the same intrahepatic bile duct tissue sample, characterized in that: The steps include: (1) Sample preparation: Obtain human intrahepatic bile duct tissue, wash it, mince it, and transfer the minced tissue pieces into a centrifuge tube; (2) Enzymatic hydrolysis: Add culture medium, add collagenase II (v / v) solution with a final concentration of 0.5%, collagenase IV solution with a final concentration of 0.3%, and hyaluronidase (v / v) solution, and perform digestion and enzymatic hydrolysis at 37°C for 15 min. After the enzymatic hydrolysis at 37°C, add 0.2% (v / v) pancreatin solution, mix well, and let stand at room temperature; (3) Separation: After standing at room temperature, a first supernatant and a first sedimented tissue pellet are obtained, and the collected first supernatant is subjected to a first centrifugation to obtain a second supernatant and a second cell pellet. The first centrifugation conditions are 2000-3000×g, 5-10 min, and 4-6°C. (IV) Exosome preparation: The second supernatant in step (3) was centrifuged again for a second time at 13,000 × g to 16,000 × g, 5 to 10 min, and 4 to 6° C. to obtain a third supernatant and a third cell pellet. After centrifugation, the third supernatant was sieved, and the filtrate was subjected to exosome purification using the Invitrogen-Total Exosome Isolation Reagent from cell culture media kit. (V) Preparation of suspension: Preparation of single cell suspension: The second cell pellet in the above step (iii) and the third cell pellet in the step (iv) were resuspended in culture medium, transferred to the same centrifuge tube, and sieving was performed; after the sieving was completed, the first centrifugation was performed, and the first centrifugation conditions were 500-800 × g, 5-10 min, 4-6 ° C. After the centrifugation was completed, the supernatant was discarded, and red blood cell lysis solution was added to the cell pellet, and the mixture was mixed by pipetting and allowed to stand at room temperature. After the red blood cell lysis was completed, the second centrifugation was performed, and the second centrifugation conditions were 500-800 × g, 5-10 min, 4-6 ° C. After the centrifugation was completed, the supernatant was discarded, and the cell pellet was resuspended by adding magnetic bead dead cell removal reagent, and incubated for 15 minutes, and then 1× building The LS column was rinsed with buffer, and the cell mixture was passed through the column and centrifuged for the third time. The conditions for the third centrifugation were 300-500×g, 5-10 min, and 4-6°C. The supernatant was discarded after the centrifugation, and the cell pellet was centrifuged for the fourth time by adding pre-cooled culture medium. The conditions for the fourth centrifugation were 100-500×g, 5-10 min, and the supernatant was discarded. The cell pellet was resuspended in pre-cooled culture medium to obtain a single cell suspension; Preparation of single cell nucleus suspension: The tissue pellet from the first sedimentation in the above step (iii) was washed with PBS, centrifuged and washed for 5-10 minutes at 500-1000 × g, and then washed again with PBS. After washing, the tissue pellet was resuspended in lysis buffer and lysed. The lysis buffer contained the following components at a final concentration of 50 mM Tris-HCl pH = 7.5, 1 mM CaCl2, 5 mM NaCl, 0.3%-0.4% (v / v) NP40, 0.3%-0.4% (v / v) Tween 20, and 0.4 U / μL RNase Inhibitor. The entire process was incubated on ice. After lysis, the tissue pellet was sieved and the filtrate was collected after sieving. The supernatant was discarded after a second centrifugation. The conditions for the second centrifugation were 800-1000 × g and 4-6°C for 5-15 minutes. The cell nucleus wash buffer was added to resuspend the cell nucleus pellet. The cell nucleus wash buffer contained the following components at a final concentration of 50 mM Tris-HCl pH = 7.5, 1mM CaCl2, 5mM NaCl, 0.4U / μL RNase Inhibitor, 1% to 2% (v / v) BSA, discard the supernatant after the third centrifugation wash, the third centrifugation conditions are 500-1000×g, 4°C centrifugation for 5-10 minutes, resuspend the cell nuclear pellet with PBS containing 1% BSA to obtain a single cell nuclear suspension.

2. The method according to claim 1, wherein In step (1), the fresh weight of the human intrahepatic bile duct tissue is 500-800 mg; and / or the number of washings is 1-4 times.

3. The method according to claim 1, wherein In step (2), the culture medium refers to RPMI 1640 culture medium with 1% BSA added.

4. The method according to claim 1, wherein In step (5), in the preparation of single cell suspension: The volume of the red blood cell lysis solution is 6 to 10 mL; and / or, the standing time is 4 to 8 min; and / or, the pre-cooled culture medium refers to RPMI 1640 culture medium with 1% BSA added, wherein the pre-cooling temperature is 4°C.

5. The method according to claim 1, wherein The total RNA amount of exosomes obtained by the method was 300 ng, and the exosome concentration was 1.06E+10; The total cell volume of the single cell suspension obtained by the method reached 98W, the cell suspension fragment rate was 4%, and the clumping rate was 4%; The total amount of cell nuclei in the single cell nucleus suspension obtained by the method reaches 220W, the fragmentation rate of the cell nucleus suspension is 5%, and the clumping rate is 5%.

6. A method for simultaneously performing small RNA sequencing, single-cell sequencing, and single-cell nucleus sequencing based on the same intrahepatic bile duct tissue sample, characterized in that: The method comprises preparing exosomes, single cell suspension and single cell nucleus suspension according to the method of claim 1, and performing small RNA sequencing, single cell sequencing and single cell nucleus sequencing.

7. A reagent or kit, characterized in that It comprises cell enzymatic solution, cell nucleus lysis solution and cell nucleus washing buffer; wherein the cell enzymatic solution is 0.5% collagenase II (v / v) solution, 0.3% collagenase IV solution, 0.1% hyaluronidase solution and 0.2% (v / v) trypsin; The cell nuclear lysis solution is a lysis buffer composed of the following components: final concentrations: 50 mM Tris-HCl pH = 7.5, 1 mM CaCl2, 5 mM NaCl, 0.3% to 0.4% (v / v) NP40, 0.3% to 0.4% (v / v) Tween 20, and 0.4 U / μL RNase Inhibitor; The cell nucleus washing buffer contains the following components with final concentrations of: 50 mM Tris-HCl pH=7.5, 1 mM CaCl2, 5 mM NaCl, 0.4 U / μL RNase Inhibitor and 1% to 2% (v / v) BSA.

8. Use of the reagent or kit according to claim 7 for simultaneously preparing exosomes, single cell suspension and single cell nucleus suspension in the same human intrahepatic bile duct tissue sample.

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