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Method Article
Paul Khavari
Stanford University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In general, an RNA-binding protein (RBP) may be considered a protein with an abnormally frequent interaction with RNA, and a "target RNA" for a specific protein may be considered an RNA with an abnormally frequent interaction with that protein. Traditional ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing analysis methods generally define interactions by methods that are indirectly assessing the latter. However, there has been limited direct assessment of these metrics by determining RNA cross-link rates or the RNA-binding profiles of non-RBPs.
Here, we describe a method to determine RNA cross-link rates and create sequencing libraries for a protein of interest, either of which may be performed on their own. easyCLIP is a relatively short, reliable, and efficient CLIP method, with the capacity to directly visualize RNA libraries and diagnose methodological problems. By combining the sequencing data and cross-link rates, the absolute frequency of given cross-links may be compared between control and experimental proteins, to nominate potential RBPs and distinctive RNA-protein interactions.
Keywords
RNA, RNA-binding protein, CLIP, Cancer
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Associated Publications
easyCLIP analysis of RNA-protein interactions incorporating absolute quantification
by Douglas F. Porter, Weili Miao, Xue Yang, +6
Nature Communications (10 March, 2021)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Paul Khavari
Stanford University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 25 Mar, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In general, an RNA-binding protein (RBP) may be considered a protein with an abnormally frequent interaction with RNA, and a "target RNA" for a specific protein may be considered an RNA with an abnormally frequent interaction with that protein. Traditional ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing analysis methods generally define interactions by methods that are indirectly assessing the latter. However, there has been limited direct assessment of these metrics by determining RNA cross-link rates or the RNA-binding profiles of non-RBPs.
Here, we describe a method to determine RNA cross-link rates and create sequencing libraries for a protein of interest, either of which may be performed on their own. easyCLIP is a relatively short, reliable, and efficient CLIP method, with the capacity to directly visualize RNA libraries and diagnose methodological problems. By combining the sequencing data and cross-link rates, the absolute frequency of given cross-links may be compared between control and experimental proteins, to nominate potential RBPs and distinctive RNA-protein interactions.
Loading...
Associated Publications
easyCLIP analysis of RNA-protein interactions incorporating absolute quantification
by Douglas F. Porter, Weili Miao, Xue Yang, +6
Nature Communications (10 March, 2021)