Executing cell-specific cross-linking immunoprecipitation and sequencing (seCLIP) in C. elegans

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA-binding proteins (RBPs) play crucial roles in gene regulation.
• Understanding RBP interaction sites is vital for deciphering gene expression control.
• Existing methods may lack efficiency or specificity for transcriptome-wide analysis.

Purpose of the Study:

• To provide a detailed protocol for cell-specific single-end enhanced cross-linking immunoprecipitation (seCLIP) in Caenorhabditis elegans.
• To enable efficient and unbiased identification of RNA-binding protein (RBP) interaction sites.
• To offer guidance on transgene construction, library preparation, sequencing, and data analysis.

Main Methods:

• Development and validation of a cell-specific seCLIP protocol.
• Utilizing Cas9-mediated chromosomal integration for transgene construction.
• Detailed procedures for RBP-associated RNA fragment isolation, library preparation, and sequencing.
• Guidelines for data analysis and interpretation.

Main Results:

• Establishment of a robust and reproducible seCLIP protocol for C. elegans.
• Demonstration of the method's capability for transcriptome-wide RBP interaction site mapping.
• Successful integration of transgenes and isolation of target RBP-bound RNAs.

Conclusions:

• The presented cell-specific seCLIP protocol is a valuable tool for studying RBP functions in C. elegans.
• This method facilitates efficient and unbiased interrogation of RBP-RNA interactions.
• The protocol supports further research into post-transcriptional gene regulation.