Low-temperature dissociation kit suitable for single cell sequencing and application thereof

A single-cell sequencing and kit technology, which is applied in the biological field, can solve the problems that protease cannot be stored for a long time and limit its wide application, and achieve the effects of simple operation, improving data quality and avoiding operation errors.

Active Publication Date: 2021-11-05
SHANGHAI OE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technical solution has not yet formed a relevant kit, and problems such as the inability of protease to preserve activity for a long time have yet to be resolved, which limits the wide application of this technical solution, and it is urgent to develop a kit to fill this gap

Method used

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  • Low-temperature dissociation kit suitable for single cell sequencing and application thereof
  • Low-temperature dissociation kit suitable for single cell sequencing and application thereof
  • Low-temperature dissociation kit suitable for single cell sequencing and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] This example describes the application of the kit of the present invention (stored at -20°C for 6 months) in single-cell sequencing of rat lung tissue. The working concentration of low-temperature protease in the kit of the present invention is 5 mg / mL, and the CaCl in enzymolysis buffer 1 2 The working concentration of DNase I was 5 mM, and the working concentration of DNase I in enzymatic digestion buffer 2 was 100 U / mL.

[0084] 1) Prepare reagents.

[0085] Before starting to prepare samples, take out the enzymatic hydrolysis solution, enzymatic hydrolysis buffer 1, and enzymatic hydrolysis buffer 2 from the -20°C refrigerator, and place them on ice until they melt.

[0086] 2) Prepare the sample.

[0087] The freshly obtained rat lung tissue was placed in a petri dish containing DMEM medium pre-cooled on ice. Rinse the tissue with pre-cooled DMEM medium on ice, suck out the rinsed medium, and then add new pre-cooled medium, repeat this process 3 times until no o...

Embodiment 2

[0105] This example describes the application of the kit of the present invention (-20°C, stored for 6 months) in single-cell sequencing of human tonsil tissue. The working concentration of low-temperature protease in the kit of the present invention is 5 mg / mL, and the CaCl in enzymolysis buffer 1 2 The working concentration of DNase I was 5 mM, and the working concentration of DNase I in enzymatic digestion buffer 2 was 100 U / mL.

[0106] 1) Prepare reagents.

[0107] Before starting to prepare samples, take out the enzymatic hydrolysis solution, enzymatic hydrolysis buffer 1, and enzymatic hydrolysis buffer 2 from the -20°C refrigerator, and place them on ice until they melt.

[0108] 2) Prepare the sample.

[0109] The freshly obtained human tonsil tissue was placed in a petri dish containing DMEM medium pre-cooled on ice. Place the petri dish on ice, rinse the tissue with DMEM medium pre-cooled on ice, suck out the rinsed medium, and then add new pre-cooled medium, repea...

Embodiment 3

[0127] This example describes the application of the kit of the present invention (stored at -20°C for 6 months) in single-cell sequencing of mouse liver tissue. The working concentration of low-temperature protease in the kit of the present invention is 5 mg / mL, and the CaCl in enzymolysis buffer 1 2 The working concentration of DNase I was 5 mM, and the working concentration of DNase I in enzymatic digestion buffer 2 was 100 U / mL.

[0128] 1) Prepare reagents.

[0129] Before starting to prepare samples, take out the enzymatic hydrolysis solution, enzymatic hydrolysis buffer 1, and enzymatic hydrolysis buffer 2 from the -20°C refrigerator, and place them on ice until they melt.

[0130] 2) Prepare the sample.

[0131] The freshly harvested mouse liver tissue was placed in a petri dish containing DMEM medium pre-cooled on ice. Place the petri dish on ice, rinse the tissue with DMEM medium pre-cooled on ice, suck out the rinsed medium, and then add new pre-cooled medium, re...

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Abstract

The invention discloses a low-temperature dissociation kit suitable for single cell sequencing. The kit comprises an enzymatic hydrolysate, an enzymatic hydrolysis buffer solution 1 and an enzymatic hydrolysis buffer solution 2. The invention also discloses an application of the kit in single cell sequencing. The low-temperature dissociation kit suitable for single cell sequencing can powerfully promote the development and wide application of low-temperature dissociation, and further improve the quality of single cell sequencing data. The components of the kit provided by the invention can be directly used, and an operator does not need to prepare a dissociation liquid; and the efficiency is improved, the operation steps are standardized, operation errors caused by manual configuration can be avoided, and the data quality is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for low-temperature dissociation of animal tissues and its application and usage method in single-cell sequencing. Background technique [0002] Single-cell sequencing technology has become a basic technical means of life science research, providing unparalleled data for people to understand the mechanism behind life phenomena. The main process of single-cell sequencing is: 1) Dissociate tissue to prepare single-cell suspension; 2) Construction of sequencing library; 3) Sequencing; 4) Data analysis. The preparation of single-cell suspension is the first and most critical step in single-cell sequencing. At present, the preparation of single-cell suspension mainly adopts the method of enzymatic digestion, and the enzymatic hydrolysis system is composed of trypsin, collagenase and other enzymes. The temperature at which these enzymes work is usually around 37°C, so d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2521/537Y02A50/30
Inventor 殷昊朱燕敏王树伟肖云平
Owner SHANGHAI OE BIOTECH CO LTD
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