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Method for separating cell nucleus from human frozen tumor tissue suitable for single cell sequencing

A tumor tissue and cell nucleus technology, applied in the field of sequencing, can solve the problems of lack of quality control results display instructions, etc., achieve the effect of simple reagent consumables and increase recovery rate

Inactive Publication Date: 2019-04-16
杭州瑞普基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 suspension separation method for frozen brain tissue through expensive auxiliary equipment, and there is no demonstration of quality control results.

Method used

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  • Method for separating cell nucleus from human frozen tumor tissue suitable for single cell sequencing
  • Method for separating cell nucleus from human frozen tumor tissue suitable for single cell sequencing

Examples

Experimental program
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Embodiment 1

[0062] (1) Reagent preparation

[0063] 1. NST lysis buffer (prepare the following two components separately and combine and mix well):

[0064] 1) 80mL of NST: 146mM NaCl, 10mM Tris base at pH 7.8, 1mM CaCl 2 ,21mMMgCl 2 ,0.05%BSA,0.2%Nonidet P-40);

[0065] 2) 20mL of 106mM MgCl 2 , 5mM EDTA, 1mg DAPI;

[0066] 2. Nuclei Wash and Resuspension Buffer (Nuclei W&R Buffer):

[0067] 1×PBS, 1.0% BSA, 0.2U / μL RNase inhibitor.

[0068] (2) Tissue cell lysis

[0069] 1. Pre-cool the prepared NST / DAPI lysis buffer and Nuclei W&R Buffer in a refrigerator at 4°C;

[0070] 2. Put the plastic petri dish on ice to pre-cool, add 3mL NST / DAPI lysis buffer;

[0071] 3. Put ~30mg of tissue into a Petri dish, cut it into pieces smaller than 1mm3 with a scalpel blade, place it on ice for 15 minutes, and shake gently every 5 minutes to mix;

[0072] 4. Disperse the tissue by blowing and aspirating 10-15 times, and filter it into a 15mL tube with a 37μm cell strainer;

[0073] (3) Nucle...

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Abstract

The invention discloses a method for separating a cell nucleus from a human frozen tumor tissue suitable for single cell sequencing and a method for extracting the cell nucleus. The method for extracting the cell nucleus comprises performing splitting treatment on a sample to be extracted in a first buffer solution; performing first centrifugation on a split product to obtain a cell nucleus precipitate; rinsing the cell nucleus and conducting resuspending treatment in a second buffer solution to obtain the cell nucleus; wherein the first buffer solution comprises: 116.8 mM of NaCl, 8 mM of Tris base at pH 7.8, 0.8 mM of CaCl2, 38 mM of MgCl2, 0.04% BSA, 0.16% Nonidet P-40, 1 mM of EDTA and 1 mg / mL of DAPI. The second buffer solution comprises 1*PBS, 1.0% BSA and 0.2U / [mu]L of RNase inhibitor. According to the method, only a simple reagent is required, no expensive separation equipment is required, and sufficient complete mononuclear can be obtained from the less frozen tumor tissue forsingle cell RNA sequencing.

Description

technical field [0001] The invention relates to the field of sequencing, in particular, the invention relates to a method for separating nuclei from human frozen tumor tissues suitable for single-cell sequencing. Background technique [0002] Single-cell sequencing refers to a new technology for high-throughput sequencing analysis of the genome, transcriptome, and epigenetic group at the level of a single cell. It is used to reveal the gene structure and gene expression status of a single cell and reflect the heterogeneity among cells. In 2013, it was rated as the annual technology by Nature Methods, and Science was listed as the top six fields worthy of attention in the year. It is the key technology applied in the 2017 Human Cell Atlas Project. Single-cell sequencing can be applied to research in developmental biology, immunology, nervous system cells, tumor tissues, etc. Among them, the use of large-scale single-cell transcriptome sequencing for tumor heterogeneity-relate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2535/122
Inventor 林锐郭成林
Owner 杭州瑞普基因科技有限公司
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