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

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…

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%