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Fusion protein used for single-cell ChIP-seq library preparation and application thereof

A technology of chip-seq and fusion protein, applied in the field of fusion protein prepared by single-cell ChIP-seq library, can solve the problems of library main information loss, high library background, high probability of experimental failure, etc., to reduce background and resolution, reduce Library background, the effect of improving the efficiency of library construction

Active Publication Date: 2019-10-25
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1. The efficiency of library construction is low, resulting in serious loss of library information when making a small number of cells
[0009] When building a library of DNA fragments released after Protein A-MNase cleavage, the traditional TruSeq library building strategy needs to be used, and the adapter and DNA fragment connection efficiency is low when building a library
Especially when a small number of cells are used as the starting material, too many DNA fragments are lost during the library construction process, which eventually leads to the loss of the main information of the library, the high probability of experimental failure, and the inability to obtain a protein-DNA interaction map at the genome-wide level
[0010] 2. Higher library background
[0012] 3. Existing ChIP-seq technology cannot perform in situ detection on tissue sections, etc., destroying its spatial resolution

Method used

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  • Fusion protein used for single-cell ChIP-seq library preparation and application thereof
  • Fusion protein used for single-cell ChIP-seq library preparation and application thereof
  • Fusion protein used for single-cell ChIP-seq library preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 5～6

[0062] The fusion protein of Examples 5-6 below is Protein A-Tn5, and the formulations of washing buffer A, washing buffer B, washing buffer C, antibody incubation buffer, etc. used are as follows (for high-throughput single cells):

[0063] Wash buffer A: 20mM HEPES, 150mM NaCl and 0.5mM spermidine;

[0064] Wash buffer B: 20 ​​mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 0.01% (w.t.) digitonin;

[0065] Wash buffer C: 20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 0.01% (w.t.) digitonin and 0.1% Triton X-100;

[0066] Activation buffer: 20mM HEPES, 10mM KCl, 1mM CaCl2, 1mM MnCl2

[0067] Antibody incubation buffer: 20mM HEPES, 150mM NaCl, 0.5mM spermidine, 0.01% (w.t.) digitonin, 0.1% Triton X-100 and 2mM EDTA;

[0068] Single-cell reaction buffer: 25 mM Mg2+, 50 mM trismethylaminopropanesulfonic acid and 0.01% (w.t.) digitonin;

[0069] Single cell stop buffer: 40 mM EDTA.

[0070] Single cell sorting buffer: 2% BSA / PBS+2mM EDTA

[0071] Single cell lysis buffer: 10mM Tris-...

Embodiment 1

[0072] Embodiment 1, the design, expression and purification of highly active ProteinA-Tn5 (PAT protein)

[0073] This embodiment provides the design and purification of an ultra-high activity Tn5 ((mutant Tn5 enhances enzyme activity) and ProteinA-Tn5 fusion protein. This method has low cost and high protein production efficiency. The method includes the following steps:

[0074] 1. Design of ProteinA-Tn5 fusion protein (PAT)

[0075] 1) Cloning the nucleic acid sequence of the mutant Tn5E54K, L372P (hereinafter referred to as Tn5) into the pET28 expression vector, such as figure 2 shown.

[0076] 2) The IgG recognition domain of 2xProtein A is connected.

[0077] 3) Sanger sequencing was used to ensure the authenticity of the coding region.

[0078] Compared with wild-type Tn5, the mutant Tn5 in PAT has stronger adapter binding ability, and its cleavage activity is increased by more than ten times. The improvement of cleavage activity is conducive to its application in v...

Embodiment 2

[0100] Embodiment 2, optimization of each functional block of PAT fusion protein

[0101] 1. Optimal screening of linker sequences

[0102] Selecting a specific linker (-GGSDDDKEF-) sequence to connect different functional fragments of PAT can protect the natural conformation of ProteinA and Tn5, thereby obtaining a fusion protein with better activity.

[0103] The method of connecting fusion proteins with different linkers and the steps of activity verification are as follows:

[0104] 1. When 2xProtein A (IgG recognition sequence) is connected to the original sequence of Tn5, different linker sequences are introduced by PCR (see Table 1, a total of 10 linker sequences).

[0105] Table 1 Different linker sequences

[0106]

[0107]

[0108] 2. Obtain the PAT connected by different linkers according to the method described in Example 1, and then perform functional identification on it, the method is as follows:

[0109] 1) Take 10 μl IgG beads, wash with 200 μl HGX bu...

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Abstract

The invention relates to a fusion protein used for single-cell ChIP-seq library preparation and application thereof. The fusion protein comprises Tn5 transposase and an Fc binding protein; a kit comprises the fusion protein and other auxiliary detection reagents; a method uses the fusion protein or the kit for ChIP-seq detection. The fusion protein, the kit and the method can improve the library building efficiency and lower the library background in the ChIP-seq detection process, so that the accuracy of the ChIP-seq detection method can be improved, and the experimental flow of ChIP-seq canbe simplified.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein used for the preparation of a single-cell ChIP-seq library and its application. Background technique [0002] With the completion of gene sequencing and the advent of the post-genome era, epigenetics has become a research hotspot in the biological field. Epigenetics mainly studies the heritable modification of DNA and related protein molecules without changing the nucleotide sequence of the gene. These modifications can be "memorized" by cells and retained during subsequent cell divisions Next, its research direction includes: one is the regulation of selective expression of gene transcription level, and the other is the regulation of gene post-transcription. At present, the hotspots of epigenetics mainly focus on the selective expression regulation of gene transcription level, especially the interaction between transcription factors and DNA, DNA methyla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/12C40B50/06C12Q1/6806C12N15/10
CPCC07K14/31C07K14/315C07K2319/20C12N9/1241C12N15/1093C12Q1/6806C40B50/06C12Q2531/113
Inventor 何爱彬余先红艾珊珊
Owner PEKING UNIV
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