Analysis of Protein-RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing

TY - CHAP

T1 - Analysis of Protein-RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing

AU - König, Julian

AU - McGlincy, Nicholas J.

AU - Ule, Jernej

PY - 2012/1/23

Y1 - 2012/1/23

N2 - Post-transcriptional regulation of gene expression is controlled by the unique composition and spatial arrangement of RNA-binding proteins (RBPs) on individual transcripts. Therefore, understanding post-transcriptional regulation requires precise and comprehensive binding site maps for RBPs. UV cross-linking and immunoprecipitation (CLIP) is a state-of-the-art technique for generating such maps on a genome-wide scale. However, data complexity is often limited and the resolution of the resulting maps is confined to approximately 30 nucleotides. This, in turn, complicates the identification of individual binding sites. We recently described individual-nucleotide resolution CLIP (iCLIP) - an approach that both increases data complexity and allows binding site detection at single-nucleotide resolution. Here, we present the latest version of our iCLIP protocol, discussing critical aspects and recent modifications.

AB - Post-transcriptional regulation of gene expression is controlled by the unique composition and spatial arrangement of RNA-binding proteins (RBPs) on individual transcripts. Therefore, understanding post-transcriptional regulation requires precise and comprehensive binding site maps for RBPs. UV cross-linking and immunoprecipitation (CLIP) is a state-of-the-art technique for generating such maps on a genome-wide scale. However, data complexity is often limited and the resolution of the resulting maps is confined to approximately 30 nucleotides. This, in turn, complicates the identification of individual binding sites. We recently described individual-nucleotide resolution CLIP (iCLIP) - an approach that both increases data complexity and allows binding site detection at single-nucleotide resolution. Here, we present the latest version of our iCLIP protocol, discussing critical aspects and recent modifications.

KW - Antibody preparation

KW - iCLIP

KW - Next-generation sequencing

KW - Protein-RNA interactions

KW - Quality controls

KW - Single-nucleotide resolution

UR - http://www.scopus.com/inward/record.url?scp=84886000056&partnerID=8YFLogxK

U2 - 10.1002/9783527644582.ch10

DO - 10.1002/9783527644582.ch10

M3 - Chapter

AN - SCOPUS:84886000056

SN - 9783527328192

SP - 153

EP - 169

BT - Tag-Based Next Generation Sequencing

PB - Wiley - VCH

ER -