CN114457020A - Single cell sorting method and application thereof - Google Patents
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Abstract
The invention provides a single cell sorting method and application thereof, and the related contents comprise sample preparation, sample loading, position calibration, single cell titration sorting and single cell full-length transcriptome library analysis; the single cell can be obtained by single cell titration sorting method, the activity is high, the requirement on the initial sample cell number is low, and the optimal sorting concentration is 105The single cell/ml can also improve the success rate of constructing a single cell full-length transcriptome library, greatly save the reagent dosage of downstream experiments, and obviously improve compared with other existing single cell sorting and single cell sequencing technologies, so that the single cell sequencing technology can be more widely applied to the research of the tumor tissue microenvironment.
Description
Technical Field
The invention belongs to the technical field of biomedicine, particularly relates to a single cell sorting method and application thereof, and particularly relates to a high-activity tumor cell obtained by using a single cell titration sorting system and application thereof in single cell sequencing.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
In recent years, single cell sequencing techniques have been successfully used to resolve the complex subpopulation structures in some normal or diseased tissues. Single cell sequencing technology allows the study of molecular differences and the reconstruction of lineage hierarchies at the single cell level, enabling related researchers to address the fundamental issues of cell type and subtype biology in complex diseases, particularly tumor microenvironments. However, single cell sequencing requires single cells with high activity (more than 80% of the activity), and the current mainstream separation method for obtaining single cells is flow cytometry sorting, wherein the cell activity is greatly damaged by sheath fluid pressure, the initial sample size is large, the operation of an instrument is complex and time-consuming, a special person is required for maintenance, and the defects of economy, time, labor cost and the like are overcome, so that the research of downstream single cell sequencing and the like is limited to a certain extent.
Limiting dilution and most microfluidic techniques follow a poisson distribution, resulting in multiple cells per location, which is inefficient and data biased. CellenONE X1 used visual feedback to ensure that there was only a single cell at each location. CellenONE X1 only selected droplets containing single cells to be sorted directly into arbitrary well plates or microwells. All cells which do not meet the set conditions are put back into the sample tube to ensure that no cells are wasted, and the cells can be sucked again to be recovered for secondary sorting. The equipment carries out real-time image analysis on each drop of sample, takes a picture and records the whole experimental process. Furthermore, CellenONE X1 is an open platform that provides for single cell sorting and nanoliter reagent loading. This versatility enables the automation of more and more single-cell library preparation work today. Moreover, nanoliter-level operation will greatly reduce the cost of reagent consumables.
The conventional cell sorting experiment uses the flow cytometer to sort the cells, which are influenced by the pressure of sheath fluid, high voltage and the like, so that the activity of the cells is low, and the cells are separatedThe number of the cells to be loaded at first is higher, and the concentration is required to be 106-107Individual cells/ml. Therefore, the application of single cell sequencing research is limited, and researchers seek a flexible sorting method which is tolerant to the number of starting cells. In addition, the situation that the flow cytometer can not retain the image of a single cell when the single cell passes through the nozzle and a plurality of cells are sorted into the same hole or a large number of cells are not sorted into the hole generally exists, so that the development of a simple, efficient and accurate sorting method for improving the construction power of a single cell library becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the problem of how to obtain high-activity single cells by a simple and efficient method, improve the success rate of constructing a single cell full-length transcriptome library and save reagents.
In view of this, the invention provides a single cell sorting method and application, the specific technical scheme is as follows:
< first aspect >
The invention provides a single cell sorting method, which comprises the following steps:
s1, sample preparation, namely dissociating the cells to be sorted into suspension, and staining the suspension by using a cell activity detection reagent to prepare cell suspension;
s2, loading a sample, starting a single cell separation system, adding the cell suspension obtained in the step S1 into the initial sample well, and placing the well plate at a collection position;
s3, calibrating the position, setting single cell titration sorting parameters, and adjusting the titration position to the central position of the bottom of the pore plate;
s4, single cell titration sorting, sucking cells by a capillary tube, observing the state of liquid drops by an imaging system, opening a sorting system Mapping option, observing the cells passing through a liquid flow, obtaining various parameters of the cells in a sample, adjusting the particle size of the sorted cells to accurately capture active cells, finally forming liquid drops with a preset volume, titrating the liquid drops into each hole of a hole plate, and removing empty holes, multi-cell holes and cells stained by an active detection reagent according to the retained picture of the single cell in the nozzle area; single cells were obtained.
In step S4, the predetermined volume is (450-600pL)0.1 μ L.
The to-be-titrated sorted cells are firstly subjected to TrypLETMExpress trypsin was dissociated into a cell suspension, filtered through a 30 μm cell strainer, and counted.
Further, step S1 specifically includes: the cells to be sorted are diluted to 1X 10 with the subculture medium5At each cell/mL, 50uL was taken and stained with a cell viability-determining reagent.
The subculture medium comprises: DMEM medium containing 10% Fetal Bovine Serum (FBS).
The cell is a tumor cell.
The cell activity detection reagent is trypan blue solution.
The single cell separation system is the CellenONE X1 system.
In step S3, the single cell titration sorting parameters are X-19, Y-40, Z-144, Voltage 76 and Pulse 49.
In step S4, the cell size is 10-40 μm.
In step S2, the well plate is a 96 well plate, and 1.5. mu.L of pre-cooled 3U RNase inhibitor-containing calcium-magnesium-free PBS solution is added to each well in advance.
< second aspect >
The invention also provides a construction method of the full-length transcriptome library, which adopts the single cells sorted by the method to carry out single-cell reverse transcription and library construction.
Applications of
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method for reverse transcription of single cells per well.
Single cell reverse transcription product use
XT DNA Library Preparation Kit and
library construction was performed by the XT DNA Library Preparation Kit method.
The application of the single cell sorting method in the research of tumor evolution mechanism, accurate cancer typing, tumor drug resistance mechanism and curative effect prediction after single cell RNA sequencing also belongs to the protection scope of the application.
Preferably, the cells to be sorted are TrypLETMExpress trypsin was dissociated into single cell suspensions, filtered through a 30 μm cell strainer, and then counted using a cell counter.
The complete medium was DMEM medium containing 10% FBS.
The trypan blue solution, concentration 100. mu.M.
Preferably, when the Cellenone X1 single cell titration separation system is started, the tray of the pore plate is precooled and moved to the starting point, the air pump is started, the particle size of the cell is adjusted to be 10-40 μm, and 10 μ L of cell suspension is sucked by the PDC and then injected to the bottom of each pore of the 96 pore plate through titration.
The pore plate is a 96 pore plate with a low skirt edge.
The method further comprises titration of the sorted single cell full-length transcriptome sequencing: and after sorting and titration are finished, carrying out single cell reverse transcription and library construction on the single cells in the holes.
Preferably, reverse transcription is used
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method.
Preferably, the library construction is based on the use of
XT DNA Library Preparation Kit and
XT DNA Library Preparation Kit method.
The inventor develops a method for preparing single cells by a single cell titration sorting system, which has high activity and good compatibilityThe cell number of the initial sample is required to be low, and the optimal sorting concentration is 105The success rate of constructing a single-cell full-length transcription group library can be improved, the reagent consumption of downstream experiments is greatly saved, and compared with other existing single-cell sorting and single-cell sequencing technologies, the method has the advantages that the single-cell sequencing technology can be more widely applied to the research of tumor tissue microenvironment.
Compared with the prior art, the invention has the beneficial effects that:
1. a novel single cell titration sorting method which is flexible and can retain images of single cells when the single cells pass through a nozzle is developed, and the single cells are prepared by adjusting XYZ axis coordinates. The single cell obtained by the method has high activity and low requirement on the number of cells of an initial sample, can improve the success rate of constructing a full-length transcription group library of the single cell, greatly saves the reagent dosage of a downstream experiment, has obvious improvement compared with other existing single cell sorting and single cell sequencing technologies, and can be more widely applied to the research on the microenvironment of tumor tissues.
2. The single cell titration sorting system CellenONE X1 is an automatic single cell sorting system based on piezoelectric acoustic technology, and is characterized in that a glass capillary-based PDC technology is used for sucking a small amount of microliter cell suspension, a piezoelectric acoustic distribution/droplet imaging technology is used, the technology provides a simple and effective method for separating and recovering single cells, tracking cell phenotype and generating clone groups, the distribution condition of each droplet can be monitored in real time in the distribution process, the single cells can be stored in a target hole with high accuracy, the whole sorting process does not involve methods of damaging the cells such as high-speed liquid flow and a high-voltage deflection electric field, and the activity of the collected single cells is very high. Individual cells can be titered on a variety of microplates (96-well, 384-well, 1536-well, 5184-well, home-made microplates).
3. The invention provides a method for preparing single cells by a single cell titration sorting system for the first time, which has high activity and low requirement on the number of cells in an initial sample, can realize the efficient recovery of single target cells from a small sample, can improve the success rate of constructing a single cell full-length transcriptome library, greatly saves the reagent consumption of downstream experiments, and has remarkable improvement compared with other existing single cell sorting and single cell sequencing technologies. In addition, the method of the invention has the advantages of stability, good reproducibility and convenient operation for researchers, thereby having good practical application value.
In conclusion, the Cellenone X1 single cell titration sorter is used for sorting single cells, has the advantages of high efficiency, high activity, simplicity in operation, improvement of the success rate of single cell full-length transcriptome library construction, reduction of downstream experiment cost and the like, and greatly accelerates the application of single cell RNA sequencing in tumor evolution mechanism research, accurate cancer typing, tumor drug resistance mechanism and curative effect prediction research.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a photograph of a single cell in the nozzle region of Cellen ONE X1 of example 1;
FIG. 2 is a schematic representation of the detection under light microscopy of single cells titrated and sorted by Cellen ONE X1 of example 1;
FIG. 3 is a schematic diagram of quality control of single cell library construction of single cell reverse transcription products from well 1 titrated by Cellen ONE X1 in example 2;
FIG. 4 is a schematic diagram of quality control of single cell library construction using the single cell reverse transcription products from well 2 titrated out by Cellen ONE X1 of example 2;
FIG. 5 is a schematic diagram of quality control of single cell library construction by the single cell reverse transcription product from well 3 titrated by Cellen ONE X1 of example 2;
FIG. 6 is a schematic diagram of quality control of single cell library construction by single cell reverse transcription products from well 4 titrated by Cellen ONE X1 of example 2.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are exemplary intended for explaining the present invention, and it is apparent that the described embodiments are a part of the embodiments of the present invention, not all of the embodiments and not to be construed as limiting the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
According to the embodiment of the invention, the invention provides a method for preparing single cells by a single cell titration sorting system for single cell full-length transcriptome sequencing research. The content involved mainly comprises five parts: firstly, preparing a sample; secondly, loading a sample; thirdly, position calibration; fourthly, single cell titration and sorting; fifthly, analyzing the whole-length transcription group library by single cell.
Example 1
The embodiment provides a single cell titration sorting system, which comprises the following steps:
1. configuration of cell activity detection reagent: trypan blue was prepared at 100 μ M in sterile PBS for use;
2. cell culture: adding a DMEM culture solution containing 10% Fetal Bovine Serum (FBS) and 1% streptomycin into a culture dish, and culturing cells in an incubator at 37 ℃;
3. sample preparation: washing adherent tumor cells grown on the culture dish with 37 deg.C pre-warmed PBS for 3 times, and washing with 0.25% TrypLETMExpress trypsin dissociates and digests adherent cells, stops digestion with complete culture medium containing 10% FBS, centrifuges for 5min at 500g room temperature, abandons supernatant, re-suspends cell sediment with complete culture medium, prepares suspension, and adjusts suspension density to 1 × 105Taking 50ul of each cell/mL for later use, adding 100 mu M trypan blue solution according to the proportion of 1:10,000 for dyeing, preparing cell suspension for later use, and immediately preparing for single cell titration and sorting;
4. sample loading: starting a CellenONE X1 single cell titration system, loading the cell suspension of the step 2 into an initial sample well, and then placing a 96-well plate to a collection position;
5. position calibration: adjusting the titration position to the central position of the bottom of the well plate: setting single cell titration sorting parameters: x-19, Y-40, Z-144, Voltage 76, Pulse 49;
6. single cell titration and sorting: sucking cells by using PDC (piezo disperse capillary), observing the state of the liquid drops by using an imaging system, obtaining parameters such as the diameter and the roundness of the cells in a sample by adjusting the imaging system, adjusting the particle size (10-40 mu m) of the sorted cells so as to accurately capture active cells, finally forming (450-600pL) liquid drops with the volume of 0.1 mu L, and titrating the liquid drops into each hole of a hole plate, wherein 1 cell per hole; the single cells were obtained by sorting by removing empty wells, multi-cell wells, and cells stained with trypan blue from the retained photographs of the single cells in the nozzle area.
Wherein the well plate is a 96 well plate, and 1.5. mu.L of precooled calcium-magnesium-free PBS solution containing 3U of RNase inhibitor is added in each well in advance.
FIG. 1 is a schematic photograph showing a single cell in the Cellen ONE X1 nozzle region of this example; FIG. 2 is a schematic diagram of the detection of single cells titrated and sorted by Cellen ONE X1 in this example under a light mirror.
Example 2
Full length transcriptome analysis of single cells selected by single cell titration with CellenONE X1 based on the method of example 1:
1. adding 1.5 mu L of PBS (phosphate buffer solution) containing 3U RNase inhibitor and free of calcium and magnesium into each well of a 96-well plate in advance, carrying out single cell titration sorting by using Cellenone X1, titrating 1 cell in each well, and removing empty wells, multi-cell wells and cells stained by trypan blue according to the retained pictures of the single cells in the nozzle area;
2. single cells from four wells in step 1 were randomly selected for full length transcriptome analysis: applications of
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method is used for reverse transcription of single cells in each well and then single cell reverse transcription products
XT DNA Library Preparation Kit and
XT DNA Library Preparation Kit method for Library construction and quality control,
the results are shown in FIGS. 3-6, where:
FIG. 3 is a schematic diagram of quality control of single cell library construction using single cell reverse transcription products from well 1 titrated by Cellen ONE X1 in example 2 of the present invention;
FIG. 4 is a schematic diagram of quality control of single cell library construction using single cell reverse transcription products from well 2 titrated by Cellen ONE X1 in example 2 of the present invention;
FIG. 5 is a schematic diagram of quality control of single cell library construction using single cell reverse transcription products from well 3 titrated by Cellen ONE X1 in example 2 of the present invention;
FIG. 6 is a schematic diagram of quality control of single cell library construction using single cell reverse transcription products from well 4 titrated by Cellen ONE X1 in example 2 of the present invention.
The above-described embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method of single cell sorting comprising the steps of:
s1, sample preparation: dissociating the cells to be sorted into suspension, and staining the suspension by using a cell activity detection reagent to prepare cell suspension;
s2, sample loading: starting the single cell separation system, adding the cell suspension obtained in the step S1 into the initial sample hole, and placing the hole plate at a collection position;
s3, position calibration: setting single cell titration sorting parameters, and adjusting the titration position to the central position of the bottom of the pore plate;
s4, single cell titration sorting: sucking cells by a capillary tube, observing the state of liquid drops by using an imaging system, opening a Mapping option of a sorting system, observing the cells passing through a liquid flow to obtain various parameters of the cells in a sample, adjusting the particle size of the sorted cells to accurately capture active cells, finally forming liquid drops with a preset volume, titrating the liquid drops into each hole of a hole plate, and removing empty holes, multi-cell holes and the cells stained by an activity detection reagent according to a retained picture of a single cell in a nozzle area to obtain single cells.
2. The single-cell sorting method of claim 1, wherein the cells are tumor cells.
3. The single-cell sorting method according to claim 1, wherein the cell activity detection reagent is trypan blue solution.
4. The single-cell sorting method of claim 1, wherein the single-cell separation system is the CellenONEX1 system.
5. The single-cell sorting method according to claim 1, wherein in step S4, the cell size is 10-40 μm.
6. The single-cell sorting method according to claim 1, wherein in step S2, the well plate is a 96-well plate, and 1.5. mu.L of pre-cooled 3U RNase inhibitor-containing calcium-magnesium-free PBS solution is added to each well in advance.
7. A method for constructing a full-length transcriptome library, comprising performing single-cell reverse transcription and library construction using the sorted cells of any one of claims 1 to 6.
8. The method of claim 7, wherein reverse transcription is performed using
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method.
9. The method of claim 7, wherein library construction is performed using
XT DNA Library Preparation Kit and
XT DNA Library Preparation Kit method.
10. The single-cell sorting method of any one of claims 1-6, after single-cell RNA sequencing, is used in the research of tumor evolution mechanism, cancer precise typing, tumor drug resistance mechanism and curative effect prediction.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109811045A (en) * | 2017-11-22 | 2019-05-28 | 深圳华大智造科技有限公司 | The construction method of high-throughput unicellular overall length transcript profile sequencing library and its application |
| CN112280828A (en) * | 2020-10-22 | 2021-01-29 | 上海交通大学医学院 | In vitro tissue cell nucleus separation method for reducing single cell amplification bias |
| CN112646701A (en) * | 2020-12-10 | 2021-04-13 | 中国科学院深圳先进技术研究院 | One-step single cell separation and distribution system |
| CN114015750A (en) * | 2021-10-18 | 2022-02-08 | 广东省农业科学院作物研究所 | High-throughput verification method for plant tissue single cell transcriptome sequencing result |
-
2022
- 2022-02-28 CN CN202210192165.8A patent/CN114457020A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109811045A (en) * | 2017-11-22 | 2019-05-28 | 深圳华大智造科技有限公司 | The construction method of high-throughput unicellular overall length transcript profile sequencing library and its application |
| CN112280828A (en) * | 2020-10-22 | 2021-01-29 | 上海交通大学医学院 | In vitro tissue cell nucleus separation method for reducing single cell amplification bias |
| CN112646701A (en) * | 2020-12-10 | 2021-04-13 | 中国科学院深圳先进技术研究院 | One-step single cell separation and distribution system |
| CN114015750A (en) * | 2021-10-18 | 2022-02-08 | 广东省农业科学院作物研究所 | High-throughput verification method for plant tissue single cell transcriptome sequencing result |
Non-Patent Citations (3)
| Title |
|---|
| VALERIA FERNANDEZ VALLONE等: "Methods for Automated Single Cell Isolation and Sub-Cloning of Human Pluripotent Stem Cells", PROTOCOL, pages 9 - 14 * |
| 周莹;黄华艺;: "单细胞测序技术及其在肿瘤研究和临床诊断中的应用", 分子诊断与治疗杂志, no. 03, pages 147 - 172 * |
| 肖政云: "单细胞转录本测序技术开发及在肝癌中的研究应用", 中国博士学位论文全文数据库 医药卫生科技辑, pages 46 * |
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