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cCLIP-Seq: Retrieval of Chimeric Reads from HITS-CLIP (CLIP-Seq) Libraries

  • Panagiotis Alexiou
  • Manolis Maragkakis
  • Zissimos Mourelatos
  • Anastassios Vourekas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)

Abstract

HITS-CLIP (High-Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation, CLIP-Seq) libraries contain fragments of the RNA sequences bound in vivo by an RNA binding protein (RBP). Such fragments, especially if they represent RNA duplexes bound in vivo by the RBP, can occasionally be ligated together to form chimeric CLIP tags. Chimeric CLIP tags from Argonaute CLIP libraries can provide the exact base pairing profiles of small RNAs with their target RNA sequences, thus solving a critical problem in the field of post-transcriptional regulation. We recently reported an analysis of chimeric reads from the Drosophila Piwi protein Aubergine, which revealed a novel mechanism for mRNA entrapment within germ RNP granules. We term this novel approach chimeric CLIP (cCLIP) and present here the main steps that a researcher can take after the acquisition of the deep sequencing data, for the identification of candidate chimeric reads in Piwi CLIP libraries. Extending the scope beyond small-RNA binding proteins, we believe that cCLIP can be utilized to elucidate the in vivo functions of RNA-binding proteins in general, and especially those that modulate RNA secondary structures. We, therefore, also describe aspects of the generalized chimeric read identification problem, which can find use in the analysis of the CLIP libraries of any RNA-binding protein.

Key words

HITS-CLIP CLIP-Seq RNA-IP Argonaute Piwi Next generation sequencing Illumina cDNA Immunoprecipitation Chimeric reads RNA-binding protein Ribonucleoprotein complexes In vivo Transcriptomic analysis Base-paired RNA miRNA target sequences piRNA target sequences Posttranscriptional RNA processing Gene silencing 

Notes

Acknowledgments

We thank members of the Mourelatos laboratory for discussions. This research was supported by NIH grant GM072777 and a grant from ALS Therapy Alliance to Z.M.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Neuropathology, Department of Pathology and Laboratory Medicine, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA

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