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Kit for separating brain and spinal cord tissue cell nucleuses and separation method of cell nucleuses

A technology of cell nuclei and kits, which is applied in the direction of vertebrate cells, biochemical equipment and methods, animal cells, etc. It can solve the problems of cumbersome flow sorting operations, high tissue volume requirements, and many cell nuclei, and achieve high integrity , low cost, and low cell activity

Pending Publication Date: 2022-04-15
BIOMARKER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to meet the requirements of single-cell nuclear transcriptome sequencing, most of the commonly used single-cell nucleus isolation methods currently require flow cytometry to purify nuclei. The requirement for the amount of tissue is higher, generally 200mg (about the size of soybeans) is required
In addition, there is currently no perfect quality control standard for the nuclei obtained by flow cytometry sorting. Sequencing directly after flow cytometry sorting may lead to poor data and requires repeated experiments.

Method used

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  • Kit for separating brain and spinal cord tissue cell nucleuses and separation method of cell nucleuses
  • Kit for separating brain and spinal cord tissue cell nucleuses and separation method of cell nucleuses
  • Kit for separating brain and spinal cord tissue cell nucleuses and separation method of cell nucleuses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Isolation of mouse hypothalamic nucleus

[0060] This embodiment uses the mouse hypothalamus tissue as the experimental material to provide a method for isolating cell nuclei. The specific steps are as follows:

[0061] (1) Take the frozen 20mg mouse hypothalamus, and immediately transfer it to a 1.5mL pre-cooled centrifuge tube; add 100 μL of lysate, time it immediately, and cut it into small pieces with pre-cooled scissors. Crumble for about 2 minutes; continue to add 400 μL of lysis solution, and lyse on ice for a total of 5 minutes (timing from the first addition of lysis solution to the end of lysis);

[0062] Among them, the formula of 500μL cell nucleus lysate is as follows: 320mM sucrose, 25mM KCl, 5mM MgCl 2 , 10mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 Substitute, 0.2U / μL Sigma Protector RNase inhibitor, 1mM DTT, DMEM to 500μL;

[0063] (2) After the lysis is completed, stop the lysis with 500 μL of cell nucleus washing solution to obtain the lysate; ...

Embodiment 2

[0068] Example 2 Isolation of mouse spinal cord nuclei

[0069] This embodiment uses mouse spinal cord tissue as an experimental material to provide a method for isolating cell nuclei. The specific steps are as follows:

[0070] (1) Take 50 mg of frozen mouse spinal cord tissue and immediately transfer it to a 1.5 mL pre-cooled centrifuge tube; add 100 μL of lysate, time it immediately, and cut it into small pieces with pre-cooled scissors. Crumble for about 2 minutes; continue to add 400 μL of lysis solution, and lyse on ice for a total of 5 minutes (timing from the first addition of lysis solution to the end of lysis);

[0071] The recipe for 500 μL of cell nucleus lysate is as follows: 320 mM sucrose, 25 mM KCl, 5 mM MgCl 2 , 10mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 Substitute, 0.2U / μL Sigma Protector RNase inhibitor, 1mM DTT, DMEM to 500μL.

[0072] (2) After the lysis is completed, stop the lysis with 500 μL of cell nucleus cleaning solution to obtain the lysate;

[00...

Embodiment 3

[0079] This embodiment uses mouse brain tissue as the experimental material to provide a method for isolating cell nuclei. The specific steps are as follows:

[0080] (1) Take the frozen 50 mg mouse brain tissue of 6 weeks, and immediately transfer it to a 1.5 mL pre-cooled centrifuge tube; add 100 μL of lysate, time it immediately, and cut it into small pieces with pre-cooled scissors, the smaller the better , cut into pieces for about 2 minutes; continue to add 400 μL of lysis solution, and lyse on ice for a total of 5 minutes (timing from the first addition of lysis solution to the end of lysis);

[0081] The recipe for 500 μL of cell nucleus lysate is as follows: 320 mM sucrose, 25 mM KCl, 5 mM MgCl 2 , 10mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 Substitute, 0.2U / μL Sigma Protector RNase inhibitor, 1mM DTT, make up to 500μL with solvent, wherein PBS is used as the solvent in scheme 1, and DMEM medium is used in scheme 2 as the solvent.

[0082] (2) After the lysis is completed...

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Abstract

The invention relates to the technical field of single cell nucleus transcriptome sequencing, in particular to a kit for separating brain and spinal cord tissue cell nucleuses and a cell nucleus separation method. The kit provided by the invention comprises a lysis solution and a cell nucleus cleaning solution. The invention also provides a method for extracting cell nucleuses by using the kit. According to the kit and the extraction method, only a small amount of tissue is needed for extraction, the mononuclear suspension with high number and quality of cell nucleuses can be obtained without complex flow cytometry sorting, the requirement of mononuclear transcriptome sequencing can be well met, high-quality transcriptome data can be obtained, and the method is suitable for large-scale popularization and application. The method has the advantages of low cost and simplicity and rapidness in operation.

Description

technical field [0001] The invention relates to the technical field of single cell nucleus transcriptome sequencing, in particular to a kit for isolating nuclei of brain and spinal cord tissue and a method for isolating nuclei. Background technique [0002] In recent years, single-cell nuclear transcriptome sequencing has attracted more and more attention because it has no requirement for cell diameter, no dissociation effect, and can truly reflect the cell types of solid tissues. When performing single-cell nuclear sequencing, it is first necessary to separate high-quality single-cell nucleus suspensions. In order to meet the requirements of single-cell nuclear transcriptome sequencing, most of the commonly used single-cell nucleus isolation methods currently require flow cytometry to purify nuclei. The requirement for the amount of tissue is relatively high, generally 200mg (about the size of soybeans) is required. In addition, there are currently no perfect quality cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12Q1/6869
Inventor 郑洪坤刘敏杨明涛王丽娟
Owner BIOMARKER TECH
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