title: Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.
abstract: 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. Hence, the accuracy and sensitivity of binding site identification is critical.
  
  
  
  We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. 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 also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks.
  
  
  
  Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.
authors.affiliation: Department of Medicine, University of Colorado Denver, Aurora, CO, USA. austin.gillen@ucdenver.edu.
authors.forename: Austin E
authors.initials: AE
authors.lastname: Gillen
authors.affiliation: Department of Medicine, University of Colorado Denver, Aurora, CO, USA.
authors.forename: Tomomi M
authors.initials: TM
authors.lastname: Yamamoto
authors.affiliation: Department of Medicine, University of Colorado Denver, Aurora, CO, USA.|Present Address: Department of Bioengineering, University of Washington, Seattle, WA, USA.
authors.forename: Enos
authors.initials: E
authors.lastname: Kline
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, USA.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Hesselberth
authors.affiliation: Department of Medicine, University of Colorado Denver, Aurora, CO, USA. peter.kabos@ucdenver.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Kabos
mesh_terms: D016477:Artifacts; D001665:Binding Sites; D017931:DNA Primers; D015870:Gene Expression; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D047468:Immunoprecipitation / Q000379:methods; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D016601:RNA-Binding Proteins; D012313:RNA
keywords: CLIP-seq; HITS-CLIP; PAR-CLIP; iCLIP; microRNA