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…
開示
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% |
