Method for analyzing unicellular inclusion

An inclusion, single-cell technology, applied in the field of single-cell sequencing, can solve the problems of high technical threshold, consumption, information deviation, etc., and achieve the effects of a wide range of analysis targets, high capture and release efficiency, and simple and convenient use.

Inactive Publication Date: 2019-06-21
XIAMEN UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The second technical problem is the amplification of micro-contents in single cells, and how to label each cell during sample preparation in order to achieve high-throughput single-cell sequencing, that is, to introduce cell codes; There are deviations in the process. How to label the information of each transcript in a single cell, that is, to introduce molecular codes, and how to integrate cellular codes and molecular codes to achieve accurate quantification of cell contents have become the methodologies of scientific researchers in recent years. focus on innovation
[0006] The third technical challenge is to realize the high-throughput one-to-one pairing of single cells and single microspheres in microwell plates. Fluorescence-activated flow cytometry, which is widely used for microparticle sorting, is not suitable for the sorting of encoded microspheres. First, the cost of encoding microspheres is expensive. Since fluorescence-activated flow sorting requires a large amount of background microspheres, this method will cause a lot of waste of reagents. Second, the sorting efficiency is low. Secondly, the single microspheres sorted are easily broken. Unable to achieve high-efficiency high-throughput single cell and single microsphere rapid one-to-one pairing
However, the limitations of conventional microfluidic chips are also very obvious. It needs to design pumps and valves insid...

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  • Method for analyzing unicellular inclusion
  • Method for analyzing unicellular inclusion
  • Method for analyzing unicellular inclusion

Examples

Experimental program
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Embodiment 1

[0053] Example 1 High-throughput single-cell transcriptome sequencing

[0054] As a preferred embodiment of the present invention, the selected cells are 3T3 and K562 cells, the cell diameter is 7-15 microns, the size of the coded microspheres used is 30-40 microns, and the single-coded microsphere pipette and fluorescence activated flow Sequencing sample preparation with formula sorting single cells is taken as an example, and the specific working process of this embodiment is introduced:

[0055] 1. Immerse the end of the chip of the portable single particle pipette with the solution inlet and outlet in the coded microsphere suspension, and use the syringe connected to the negative pressure port to generate negative pressure to absorb the sample of the coded microsphere suspension;

[0056] 2. Transfer the portable single-particle pipette to the cleaning solution, generate negative pressure through the syringe connected to the negative pressure port again, and draw the clean...

Embodiment 2

[0074] Example 2 Transcriptome sequencing analysis of rare cells

[0075] Taking LnCap cells as an example, the cell diameter is 18-25 microns, and the specific process of Example 2 is introduced:

[0076] 1. After preparing the single microsphere microwell plate in the same process as in Example 1, prepare for the addition of single cells.

[0077] 2. Repeat the operation from step 2 to step 3 in Example 1, change the microsphere suspension to cell suspension, and add a single cell to the 96 or 384-well plate with a single particle pipette to realize a pair of cells and microspheres a pair.

[0078] 3. Add cell lysate to the 96-well plate or 384-well plate containing single cells and single microspheres, centrifuge at room temperature, and incubate.

[0079] 4. Repeat steps 5 to 9 in Example 1.

[0080] The single particle pipette developed by the present invention has extremely high single microsphere capture and release efficiency, and can directly add a single coded mic...

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Abstract

The invention relates to a method for analyzing a unicellular inclusion. The method comprises main steps of 1, by means of a portable single-particle pipettor, preparing a 96 or 384 orifice plate which loads a single coding microball; 2, sorting a unicell by means of a fluorescent activation flow sorting instrument, or separating a rare cell to a micro-orifice plate by means of the pipettor, thereby realizing quick one-to-one pairing between a unicell and a single microball; and 3, converting the inclusion information in the unicell to DNA sequence information by means of the coding microball,and analyzing the sequencing data by means of high-flux sequencing technology and bioinformatics. The method of the invention can realize high-efficiency stable low-cost separation of the single coding microball and has advantages of low technical threshold, effective overcoming of Poisson distribution, wide target range, controllable flux, low cost, etc. Furthermore the developed single-particlepipettor can be used for capturing and sequencing of the rare cell and can be widely used for the fields such as basic research and clinical diagnosis unicell analysis.

Description

technical field [0001] The invention belongs to the field of single-cell sequencing, in particular to a method for analyzing single-cell content. Background technique [0002] The heterogeneity of cells is a ubiquitous life phenomenon. As an independent living entity, the properties and differences displayed by a single cell play a vital role in the development of the entire living system. Every tissue and organ in the human body contains a variety of cell types, and each type of cell will change with the life activity of the organism. If thousands of single cells are studied, it will be blurred Therefore, it is extremely important to understand the working principles of complex organisms, the life functions and immune responses of each cell type to reveal the working mechanism of human tissues and organs and the laws of gene regulation. For example, malignant tumors that cause human cancer are highly heterogeneous tissues, composed of tumor cells of various phenotypes, and...

Claims

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Application Information

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IPC IPC(8): G16B25/00C12Q1/6869
Inventor 杨朝勇田恬陈映汶李星锐毕云鹏朱志
Owner XIAMEN UNIV
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