Isotope-free mapping of protein-RNA interactions at single-nucleotide resolution by iCLIP3

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is crucial for mapping RNA-binding protein (RBP)-RNA interactions across the transcriptome.
• Existing iCLIP protocols can be resource-intensive and require substantial input material.

Purpose of the Study:

• To introduce iCLIP version 3 (iCLIP3), a streamlined and optimized protocol for iCLIP library preparation.
• To enable high-quality transcriptome-wide mapping of RBP-RNA interactions using low-input biological samples.
• To provide a comprehensive bioinformatics workflow for analyzing iCLIP sequencing data.

Main Methods:

• Development of iCLIP version 3 (iCLIP3) protocol.
• Incorporation of near-infrared visualization for RBP-RNA complexes.
• Utilizing silica column-based RNA isolation.
• Implementation of unique dual indexing with TruSeq adapters for cost-effective multiplexing.
• Establishment of a complete bioinformatics pipeline for data analysis.

Main Results:

• iCLIP3 protocol is optimized for generating high-quality iCLIP libraries from low-input material.
• The protocol facilitates cost-effective multiplexing and sequencing through unique dual indexing.
• A detailed bioinformatics workflow is provided for identifying crosslinking events and RBP binding sites.

Conclusions:

• iCLIP3 offers a streamlined and efficient approach for transcriptome-wide RBP-RNA interaction mapping.
• The optimized protocol and bioinformatics workflow enhance the accessibility and quality of iCLIP data generation.
• This advancement supports broader research into RBP functions and their roles in gene regulation.