Single cell sorting method and application thereof

A single-cell sorting technology, applied in the field of biomedicine, can solve problems such as inability to retain, and achieve the effects of saving reagent consumption, improving the success rate of construction, and requiring high active cell counts

Pending Publication Date: 2022-05-10
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, flow cytometry sorting cannot retain the image of a single cell passing through the nozzle, and it is common to sort multiple cells into the same well or a large number of unsorted wells, which makes it easy to develop a simple, efficient and accurate sorting method. The selection of methods to improve the success rate of single-cell library construction has become an urgent problem to be solved

Method used

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  • Single cell sorting method and application thereof
  • Single cell sorting method and application thereof
  • Single cell sorting method and application thereof

Examples

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Embodiment 1

[0054] This embodiment provides a single cell titration sorting system, comprising the following steps:

[0055] 1. Configuration of cell viability detection reagents: Use sterile PBS to configure trypan blue to 100 μM for use;

[0056] 2. Cell culture: add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin DMEM culture solution to the culture dish and carry out cell culture in a 37°C incubator;

[0057] 3. Sample preparation: wash the tumor cells growing on the culture dish with 37°C pre-warmed PBS for 3 times, and wash with 0.25% TrypLE TM Express trypsin dissociates and digests adherent cells, terminates the digestion with complete culture medium containing 10% FBS, centrifuges at 500 g for 5 minutes at room temperature, discards the supernatant, resuspends the cell pellet with complete culture medium, prepares a suspension, and adjusts the suspension density to 1×10 5 Each cell / mL, take 50ul for use, add 100μM trypan blue solution at a ratio of 1:10,000 for sta...

Embodiment 2

[0064] Based on the method in Example 1, the single cells selected by CellenONE X1 single-cell titration were subjected to full-length transcriptome analysis:

[0065] 1. Add 1.5 μL calcium-magnesium-free PBS solution containing 3U RNase inhibitor to each well of a 96-well plate in advance, use CellenONE X1 to perform single-cell titration sorting, titrate 1 cell per well, and select according to the remaining nozzle area Photos of single cells excluding empty wells, multicellular wells and cells stained with trypan blue;

[0066] 2. Randomly select individual cells from the four wells in step 1 for full-length transcriptome analysis: apply The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method performs reverse transcription on single cells in each well and then uses XT DNA Library Preparation Kit and XT DNA Library Preparation Kit method for library construction and quality inspection,

[0067] The result is as Figure 3-6 shown, where:

[0068] image 3 Sche...

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Abstract

The invention provides a single cell sorting method and application thereof. The related content comprises sample preparation, sample loading, position calibration, single cell titration sorting and full-length transcriptome library analysis of single cells. Single cells can be obtained through a single cell titration sorting method, the activity is high, the requirement for the number of initial sample cells is low, the optimal sorting concentration is 105 cells/ml, the construction success rate of a single cell full-length transcriptome library can be increased, and the reagent dosage of downstream experiments is greatly saved; compared with other existing single cell sorting and single cell sequencing technologies, the single cell sorting and sequencing technology is remarkably improved, so that the single cell sequencing technology can be more widely applied to research of tumor tissue microenvironment.

Description

[0001] field of invention [0002] The invention belongs to the technical field of biomedicine, and specifically relates to a single cell sorting method and its application, in particular to a single cell titration sorting system to obtain highly active tumor cells and its application in single cell sequencing. Background technique [0003] The information disclosed in the Background of the Invention is only intended to increase the understanding of the general background of the invention, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art. [0004] In recent years, single-cell sequencing technology has been successfully used to resolve complex subpopulation structures in some normal or diseased tissues. Single-cell sequencing technology allows the study of molecular differences and reconstruction of lineage hierarchies at the level of individual cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C40B50/06G01N15/14C12Q1/6869
CPCC12N5/0693C40B50/06G01N15/1475C12Q1/6869C12N2509/00G01N2015/1006G01N2015/149G01N2015/1481
Inventor 王慧何牮李沚璠孟梅周献超
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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