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Method for separating dental embryo single cells and application thereof

A single-cell, single-cell sequencing technology, applied in the field of cell biology, to reduce difficulty, save digestion time, and preserve the original characteristics of cells

Pending Publication Date: 2020-05-26
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, which method is more suitable for the study of single-cell transcriptome sequencing of tooth germ tissue needs further research

Method used

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  • Method for separating dental embryo single cells and application thereof
  • Method for separating dental embryo single cells and application thereof
  • Method for separating dental embryo single cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Taking the 14.5-day mouse embryo as an example, the specific implementation process of the present invention will be described in detail.

[0050] ICR (Institute of Cancer Research) adult mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and were raised in the SPF-level experimental animal area of ​​the Animal Experiment Center of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. Cage thrombus was used to calculate pregnancy period. Then select 14.5-day-pregnant mice, take out the embryos under aseptic conditions after euthanasia, separate the uterine wall, amniotic membrane, and placenta one by one, and eliminate embryos with large individual differences (the whole process is operated in DPBS buffer, DPBS buffer pre- cold, try to operate on ice to prevent apoptosis). After a single mouse embryo was isolated, the dissecting dish containing the mouse embryo was transferred to a dissecting microscope, and t...

Embodiment 2

[0074] The single cells obtained in Example 1 were subjected to combined digestion with collagenase, trypsin and DNA hydrolase for sequencing using the 10x Genonics single-cell sequencing platform. According to the instructions of 10x Genonics single cell 3'reagentkits v2, the single cell library construction and sequencing obtained in the example were carried out. The obtained sequencing information is shown in Table 2 below:

[0075] Table 2 Capture efficiency information of isolated single cells for single cell sequencing

[0076]

[0077] The concentration of viable cells and the number of target cells in Table 2 represent the sample loading information during cell capture. The concentration of living cells refers to: after the tissue is digested into a single cell suspension, the cells are counted by the trypan blue staining method. Since the trypan blue cannot penetrate the cell membrane of living cells, the number of cells counted is the number of living cells, and t...

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Abstract

The invention relates to a method for separating single cells, in particular to a method for separating dental embryo tissue single cells. The method comprises the following steps: sequentially utilizing collagenase, trypsin and DNA hydrolase to digest a dental embryo tissue to obtain the dental embryo single cell; the method provided by the invention is simple to operate and short in time consumption; the damage to cells is small, and the cells can maintain original characteristics to the maximum extent; and the obtained single-cell suspension is free of adhesion and meets the single-cell sequencing requirement.

Description

technical field [0001] The present invention relates to the field of cell biology. Specifically, the present invention relates to a single cell separation method. More specifically, the present invention relates to a tooth germ tissue single cell separation method suitable for single cell transcriptome sequencing. Background technique [0002] As we all know, human beings are plagued by tooth loss due to disease factors such as caries, periodontal disease, trauma and tumors. Tooth loss causes physiological and psychological dysfunction such as chewing and pronunciation. According to statistics, before the age of 50, the average loss of teeth per person in the United States reaches 8, and the rate of tooth loss before the age of 65 reaches 35%, while the rate in China is 40%, that is, about 500 million people in the country suffer from different degrees of teeth. missing. Traditional tooth restoration methods cannot restore the basic physiological functions of patients' tee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12Q1/6806C12Q1/6869
CPCC12N5/0654C12Q1/6806C12Q1/6869C12N2509/00
Inventor 裴端卿蔡景蕾赵艺帆王曜峰陈树彬陈小明
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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