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These techniques have made important contributions to the understanding of the function of a range of RNA-binding proteins, from the microRNA-related argonaute (AGO) to other regulatory factors, including FUS and PTBPs. The advent of HITS-CLIP and its derivative techniques has facilitated the mapping of RNA-protein interactions genome-wide in a variety of tissues and organisms. Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Hence, the accuracy and sensitivity of binding site identification is critical. PMID 21633356.High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. "Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data". ^ Zhang, Chaolin Darnell, Robert B (1 June 2011)."iCLIP: protein-RNA interactions at nucleotide resolution". ^ Huppertz, Ina Attig, Jan D'Ambrogio, Andrea Easton, Laura E Sibley, Christopher R Sugimoto, Yoichiro Tajnik, Mojca König, Julian Ule, Jernej (February 2014)."Global protein-RNA interaction mapping at single nucleotide resolution by iCLIP-seq". ^ Yao, Chengguo Weng, Lingjie Shi, Yongsheng (20 February 2014)."Protein–RNA interactions: new genomic technologies and perspectives". ^ König, Julian Zarnack, Kathi Luscombe, Nicholas M.This helps to identify PCR over-amplification effects in the high-throughput sequencing step and therefore improves the quantification of binding events. The primer used for reverse transcription can contain a random sequence, which can be used to barcode cDNAs. The small amount of resulting cDNAs is then PCR amplified and sequenced using a next-generation sequencing platform. Analogous resolution can be obtained with standard HITS-CLIP methods, using the observation that RT that does read through the cross link site has a high error rate, which can be used to deduce the position of the crosslink ("Crosslink induced mutation site" (CIMS) analysis ). The RNA is then reverse transcribed, causing cDNAs to often truncate at the crosslink site, which is the key insight and unique feature in the development of iCLIP, as it allows identification of the site of RNA-protein interaction at high resolution. The radiolabelled protein-RNA complexes are then excised from nitrocellulose, and treated with proteinase to release the RNA, leaving one or two amino acids at the crosslink site of the RNA. As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes, using immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. This cross-linking step has generally less background than RNA immunoprecipitation protocols. The method uses UV light to covalently bind proteins and RNA molecules. ICLIP (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation) is a method used for identifying protein-RNA interactions. Not to be confused with Intramembrane protease, sometimes abbreviated I-CLiP.