An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

Published: February 29, 2024

Abstract

Source: Xu, Q., et al. Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis. J. Vis. Exp. (2019)

This video demonstrates the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying RNA-binding proteins in mouse testis. eCLIP enables the precise identification of binding sites where RNA-binding proteins (RBPs) interact with their target RNAs.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Tissue Harvesting and UV Crosslinking Euthanize 2 adult mice using carbon dioxide (CO2) for 1-2 min or until breathing stops. Next, perform cervical dislocation on each mouse. Harvest about 100 mg of testes from mice of appropriate age (one adult testis in this study) for each immun…

Divulgazioni

The authors have nothing to disclose.

Materials

Antibodies
Anti-mouse MOV10 antibody Proteintech, China 10370-1-AP
Anti-mouse MOV10L1 antibody Zheng et al.20109 polyclonal antisera UP2175
HRP Goat Anti-Rabbit IgG ABclonal AS014
Rabbit IgG Beyotime, China A7016
Equipment Eppendorf, Hamburg, Germany 5242R
Digital sonifier BRANSON,USA BBV12081048A
DynaMag-2 Magnet Invitrogen,USA 12321D
UV-light cross-linker UVP, USA CL-1000
Materials
Tubes Corning, USA 430791
Microtubes tubes AXYGEN , USA MCT-150-C 1.5 mL
Reagents
Acid phenol/chloroform/isoamyl alcohol Solarbio, China P1011 25:24:01
AffinityScript Enzyme Agilent, USA 600107
RNase I Invitrogen, USA AM2295
RNase Inhibitor Promega, USA N251B
RQ1 DNase Promega,USA M610A
Sample Reducing Agent Invitrogen,USA NP0009
SDS Solution Invitrogen, USA 15553027 10%

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Citazione di questo articolo
An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes. J. Vis. Exp. (Pending Publication), e21984, doi: (2024).

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