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

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.