They are involved in RNA synthesis, stability. T3 - Methods in molecular biology (Clifton, N.J. RNA-binding proteins (RBPs) play a key role in the life-time of RNAs. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries. In iCLIP, specific crosslinked RNA-protein complexes are immunoprecipitated. This approach includes additional steps to digest the proteins after crosslinking and to map the crosslink sites with reverse transcriptase. RIP-seq (RNA immunoprecipitation), iCLIP (individual nucleotide. iCLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. We used the comprehensive compendium of lncRNAs available from GENCODE. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. The labeled areas of each lane were used for iCLIP library preparation and sequencing (see Methods). Figure 1d shows the autoradiogram of the nitrocellulose membrane with all transferred, crosslinked protein-RNA complexes. solfataricus P2 cells grown to early stationary phase (Fig. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.ĪB - RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. The iCLIP experiment was performed with S. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. N2 - RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. T1 - Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein-RNA interactions
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