Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein-RNA interactions

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

Abstract

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. 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. 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.

Original languageEnglish
Title of host publicationRNA Detection
Subtitle of host publicationMethods and Protocols
EditorsImre Gaspar
PublisherSpringer
Pages427-454
Number of pages28
Volume1649
ISBN (Electronic)978-1-4939-7213-5
ISBN (Print)978-1-4939-7212-8
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Keywords

  • animals
  • cross-linking Reagents
  • DNA
  • gene library
  • humans
  • Immunoprecipitation
  • Microspheres
  • Nucleotides
  • Polymerase Chain Reaction
  • RNA
  • RNA-Binding Proteins
  • Reverse Transcription
  • Ultraviolet Rays
  • isolation & purification
  • metabolism

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