A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq

Methods Mol Biol. 2022:2404:189-205. doi: 10.1007/978-1-0716-1851-6_10.

Abstract

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.

Keywords: Enhanced individual-nucleotide crosslinking and Immunoprecipitation; Next-generation sequencing; RBP binding-site detection; RNA-binding proteins; eCLIP; iCLIP; seCLIP.

MeSH terms

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

Substances

  • RNA-Binding Proteins
  • RNA