hide
Free keywords:
Binding Sites
DNA, Circular/chemistry/genetics
Gene Expression Regulation
*Gene Library
HeLa Cells
High-Throughput Nucleotide Sequencing
Humans
Immunoprecipitation/*methods
Protein Binding
RNA/*chemistry/genetics
RNA-Binding Proteins/*chemistry/genetics
Ultraviolet Rays
High-throughput sequencing
Post-transcriptional regulation
Protein-RNA interaction
Rna
RNA-binding protein
UV crosslinking and immunoprecipitation (CLIP)
iCLIP
Abstract:
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
