In-vitro tissue cell nucleus separation method for reducing unicellular amplification bias

A cell nucleus and in vitro technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of no quality control result display and instructions, and achieve the effect of saving experimental time, maintaining cell activity, and high simplicity

Pending Publication Date: 2021-01-29
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no mature and stable method to obtain mononuclear suspension from frozen tissue. At present, there is only a mononuclear ...

Method used

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  • In-vitro tissue cell nucleus separation method for reducing unicellular amplification bias
  • In-vitro tissue cell nucleus separation method for reducing unicellular amplification bias
  • In-vitro tissue cell nucleus separation method for reducing unicellular amplification bias

Examples

Experimental program
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Embodiment 1

[0090] (1) Reagent preparation

[0091] 1.WB: 10-20mM Tris-HCl (pH 7.5-7.8), 10-20mM NaCl, 50-100mM KCl, 2-10mM MgCl 2 , 5-15mM CaCl 2 , 0.04% BSA, 1mg / mL PI and 0.2U / μL RNase inhibitor;

[0092] 2.LB: add 2uL NP-40 to 1mL of WB;

[0093] 3. D-Hanks buffer containing 1% FBS, 1 mM DTT.

[0094] (2) Tissue cell lysis

[0095] 1. Pre-cool PBS and prepared WB, LB and NB;

[0096] 2. Pre-cool the plastic petri dish, centrifuge tube, etc. on ice, add 4mL pre-cooled PBS to the petri dish to clean the tissue, and the tissue block should not exceed 3mg;

[0097] 3. Put ~3mg tissue into a pre-cooled centrifuge tube, grind it on ice with a pre-cooled grinding pestle to 0.2mm3 pieces, and place it on ice for 5-10 minutes;

[0098] 4. Blow and suck 10 to 15 times to break up the tissue, and filter it into the flow tube with a 70 μm cell filter;

[0099] (3) Nucleus cleaning

[0100] 1. Centrifuge the nuclear suspension at 500g at 4°C for 5min;

[0101] 2. Remove the supernatant ca...

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Abstract

The invention discloses a single cell sequencing cell nucleus separation method capable of reducing amplification bias and application thereof. The method comprises the following steps: carrying out cracking treatment on a sample to be extracted in a buffer solution II; carrying out primary centrifugal treatment on the cracking product to obtain a cell nucleus precipitate; and washing the cell nucleus precipitate, re-suspending the cell nucleus precipitate in a buffer solution I, carrying out secondary centrifugal treatment, and finally re-suspending and uniformly mixing the cell nucleus precipitate in a buffer solution III to obtain the finally required cell nucleus. According to the method, only conventional and simple reagent consumables are needed, expensive separation equipment, long-time gradient centrifugation and other operations are not needed, and enough and complete single cell nucleuses can be obtained from few tissue samples or cells to be used for single-cell whole-genomeexperimental research. Compared with a method that usesnucleuses processed by a commercial kit, the amplification homogeneity of subsequent whole genome amplification product is better, the amplification effect is similar to that of a complete single cell, and meanwhile time and cost are saved.

Description

technical field [0001] The invention relates to the field of gene sequencing, and the invention relates to a method for isolating cell nuclei from ex vivo tissues suitable for reducing the bias of single cell amplification. Background technique [0002] Single-cell sequencing refers to a new technology for high-throughput sequencing analysis of genome, transcriptome, epigenome and other multi-omics at the level of a single cell. The application of improved technology makes it possible to explain the gene structure and gene expression status of individual cells, and to interpret the heterogeneity among cells. Single-cell sequencing can be widely applied to research in developmental biology, immunity, tumors, etc., and a large number of high-quality research and scientific research results have emerged. Among them, single-cell whole-genome sequencing amplifies the whole genome of the DNA of a single cell obtained after dissociation from tumor tissue, and then constructs and s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2523/32C12Q2527/125C12Q2535/122
Inventor 王慧何牮孟梅刘宁宁
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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