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Method for obtaining target RNA of target RNA binding protein

A technology for binding proteins and fusion proteins, which is applied in the field of obtaining target RNA binding proteins and target RNAs, can solve the problems of non-in situ detection, large amount of cells, complicated steps, etc., to avoid the loss of many fragments, improve the quality of the library, and the method is fast simple effect

Active Publication Date: 2021-12-17
BIOISLAND LAB +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] For CLIP-seq and its derivative methods, the main defects include: 1. The amount of cells used is large; 2. Not in situ detection; 3. Low cross-linking efficiency; 4. , The steps are cumbersome, the process takes a long time, and it is easy to fail; 5. The efficiency is low and the sequencing volume is large
[0006] The CUT&RUN method requires fewer samples and is suitable for studying rare cell populations, but it has not been applied in the field of RBP-RNA interaction

Method used

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  • Method for obtaining target RNA of target RNA binding protein
  • Method for obtaining target RNA of target RNA binding protein
  • Method for obtaining target RNA of target RNA binding protein

Examples

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Embodiment

[0089] (1) Collect 100,000 mouse embryonic stem cells cultured in vitro, wash them once with PBS (PH=7.2), centrifuge at 600g room temperature for 3min, remove the supernatant, and wash once with 200μL wash buffer;

[0090] (2) 8 μL Con A beads (Bangslabs) were washed with binding buffer and mixed with the cells in (1) and incubated for 10 min, centrifuged at 100 g at 4°C, discarded the supernatant (magnetic stand), and washed with wash buffer. Add 1 μg DDX5 protein antibody to 100 μL washbuffer, 0.5 μL 5% Digitonin (digitonin), 1 μL protease inhibitor (100×), incubate at 4°C for 2-12 hours;

[0091] (3) Add 0.07 μg / μL fusion protein and incubate at 4°C for 2 hours;

[0092] The DNA coding sequence of the 6×His tag is placed at the end of the DNA coding sequence of the fusion protein precursor through plasmid construction. Introduce the expression plasmid into expression strains such as BL21 or DE3, and purify the fusion protein to obtain the tagged protein. The fusion prote...

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Abstract

The invention relates to a method for obtaining target RNA of a target RNA binding protein. The method comprises the following steps: a) contacting a permeabilized cell with an antibody which specifically recognizes the target RNA binding protein (RBP) and a fusion protein to form a fusion protein-antibody-RBP-RNA compound in the permeabilized cell; the fusion protein has a protein A and / or protein G domain, a micrococcus nuclease domain and a partner domain; b) activating the micrococcus nuclease domain for enzyme digestion; c) co-incubating the enzyme digestion product with a solid phase carrier, wherein the solid phase carrier is capable of specifically binding to the partner domain to capture the compound; and d) washing off uncombined impurities, and extracting RNA in the compound.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for obtaining target RNA of a target RNA binding protein. Background technique [0002] RNA binding protein (RBP) is involved in the production, shearing, degradation and function of RNA. Therefore, the detection of RBP-RNA interaction is crucial for the elucidation of RBP function and the understanding of RNA function, which is also a very important part of RNA biology. [0003] At present, most of the methods for detecting the interaction between RNA-binding proteins and RNA are based on UV crosslinking-immunoprecipitation combined with high-throughput sequencing (crosslinking-immunprecipitation and high-throughput sequencing, CLIP-seq) and its derivative methods. CLIP-seq is currently the most widely used high-throughput sequencing analysis technology for detecting RBP-RNA interactions, and has been used to target the interaction between RBP and RNAs at the whole cell le...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804C12Q1/6806C40B50/06
CPCC12Q1/6804C12Q1/6806C40B50/06C12Q2521/30C12Q2563/131C12Q2563/149C12Q2563/143
Inventor 姚红杰李尧益李清王新秀
Owner BIOISLAND LAB
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