Deconvolution of in vivo protein-RNA contacts using fractionated eCLIP-seq

STAR Protoc. 2022 Dec 16;3(4):101823. doi: 10.1016/j.xpro.2022.101823. Epub 2022 Nov 16.

Abstract

Thousands of RNA-binding proteins orchestrate RNA processing and altered protein-RNA interactions frequently lead to disease. Here, we present experimental and computational analysis pipelines of fractionated eCLIP-seq (freCLIP-seq), a modification of enhanced UV-crosslinking and RNA immunoprecipitation followed by sequencing. FreCLIP-seq allows transcriptome-wide analysis of protein-RNA interactions at single-nucleotide level and provides an additional level of resolution by isolating binding signals of individual RNA-binding proteins within a multicomponent complex. Binding occupancy can be inferred from read counts and crosslinking events. For complete details on the use and execution of this protocol, please refer to Biancon et al. (2022).

Keywords: Bioinformatics; Cell Biology; Molecular Biology; Protein Biochemistry; Protein expression and purification; RNAseq; Sequencing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • High-Throughput Nucleotide Sequencing / methods
  • RNA* / genetics
  • RNA* / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Transcriptome* / genetics

Substances

  • RNA
  • RNA-Binding Proteins