Library building method for accessibility of unicellular chromatin

A chromatin, single-cell technology, applied in the field of molecular biology, to achieve the effect of wide applicability

Pending Publication Date: 2020-11-27
HANGZHOU HANGENE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, there is a lack of a technology that can combine single-cell ATAC-seq seq...

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  • Library building method for accessibility of unicellular chromatin
  • Library building method for accessibility of unicellular chromatin
  • Library building method for accessibility of unicellular chromatin

Examples

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Embodiment Construction

[0030] In order to understand the technical content of the present invention more clearly, the following examples are given in detail. Referring to the accompanying drawings, the present invention will be more fully described and illustrated with exemplary embodiments of the present invention, but it does not mean that the present invention is limited thereto.

[0031] In the present invention, PerCP-Cy5.5-anti-CD45, PE-anti-CD8a, APC-Cy7-anti-CD4 antibodies were purchased from Biolegend.

[0032] Ad1 primers and Ad2 primers are common terms for ATAC-seq library construction and amplification, which are common knowledge in the field, so they will not be repeated here.

[0033] In one embodiment, we use mouse thymocytes as test samples for the purpose of studying T cells in the mouse thymus, and further enrich the double-negative T cells whose content is less than 3%.

[0034] see figure 1 , Alexa Fluor 488-labeled linker oligonucleotides were synthesized by Sangon Biotech: ...

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Abstract

The invention relates to a library building method for accessibility of unicellular chromatin. The method comprises the following steps of: designing a pair of oligonucleotide chains capable of beingcoupled fluorescently, preparing a fluorescent transposon by combining with Tn5 transposase, dissociating single-cell suspension, adding a fluorescent antibody aiming at specific protein of a target population for incubation, carrying out fluorescence labeling, sorting and enriching, carrying out PCR library building, and carrying out library sequencing. According to the library building method for the accessibility of unicellular chromatin provided by the invention, a target cell population can be screened under the condition of ensuring the activity in a cell body; a set of oligonucleotide chains capable of realizing fluorescence coupling is designed and is combined with Tn5 transposase to prepare the fluorescence transposon, quantitative operation is carried out, the applicability is wide, and the method is especially suitable for constructing tissues with few species or tissues and poorer preservation states; and the method is suitable for cell populations with extremely small proportions.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and more specifically relates to a method for building a single-cell chromatin accessibility library. Background technique [0002] The nucleosome is the basic structural unit of eukaryotic chromatin, composed of DNA and histones. Each nucleosome is formed by winding 146bp DNA on octamer histone, and the two nucleosomes are connected by a piece of connecting DNA, and the combination of DNA and histone can change dynamically. DNA wrapped around histones is not easy to bind to other proteins, and these DNAs are usually in a state of expression repression. Open DNA regions without nucleosome binding are easy to bind to regulatory proteins, which can make their downstream genes active. Cells regulate the expression of genes by changing the binding positions of DNA and histones. Therefore, obtaining the DNA sequence in an open state can be used to study the regulatory mechanism of gene ex...

Claims

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Application Information

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IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 方靖文瞿昆李杨朱连邦
Owner HANGZHOU HANGENE BIOTECH CO LTD
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