We are analyzing https://link.springer.com/article/10.1186/s12864-016-2675-5.

Title:
Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts | BMC Genomics
Description:
 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.
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Keywords {🔍}

primer, pubmed, hitsclip, article, adaptor, reverse, mispriming, google, scholar, peaks, cas, peak, sequence, fig, transcription, pcr, central, nested, rna, sequences, blue, samples, data, library, bases, overrepresentation, libraries, cells, protocol, artifacts, binding, reads, line, protected, artifact, interactions, cell, crosslinking, sites, iclip, site, results, mirna, target, emerald, pgr, proteins, resulting, original, published,

Topics {✒️}

p38 mapk/mk2-mediated induction rna-protein interactions genome-wide large-scale clip-seq data iclip--transcriptome-wide mapping micro-rna-mediated translation repression identify transcriptome-wide transcriptome-wide regulation open access charge individual-nucleotide resolution cross-linking high-throughput sequencing reads dgcr8 hits-clip reveals c-myc-mir-34b interaction vivo protein-rna interactions protein-rna interaction networks article download pdf mir-888-3p represses expression mirna-target interactions inferred identify mirna-target interactions considered genome-wide measurements hrp-conjugated secondary antibody long pre-mrna depletion hits-clip hits-clip published hits-clip datasets target mrna/protein expression weaker ago2-mrna interactions xhoi-noti restriction site microrna-related argonaute reported mirna-mrna interactions article published online published hits-clip libraries mir mimic/control mimic high confidence measurements genome-wide identification produce pcr-competent templates perfect reverse-complementary match t4 rna ligase original hits-clip protocol 2-mediated alternative splicing artifactual hits-clip data artifactual hits-clip peak rna-protein interactions protein-rna interactions state key laboratory genome-wide mapping photoactivatable-ribonucleoside-enhanced crosslinking rna-binding proteins rna binding proteins downstream functional assays common adaptor/primer pairs early hits-clip samples

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Schema {🗺️}

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         headline:Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts
         description: 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.
         datePublished:2016-05-05T00:00:00Z
         dateModified:2016-05-05T00:00:00Z
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            iCLIP
            PAR-CLIP
            Life Sciences
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            Proteomics
            Animal Genetics and Genomics
            Microbial Genetics and Genomics
            Plant Genetics and Genomics
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      headline:Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts
      description: 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.
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         microRNA
         HITS-CLIP
         CLIP-seq
         iCLIP
         PAR-CLIP
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         general
         Microarrays
         Proteomics
         Animal Genetics and Genomics
         Microbial Genetics and Genomics
         Plant Genetics and Genomics
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      name:Tomomi M. Yamamoto
      affiliation:
            name:University of Colorado Denver
            address:
               name:Department of Medicine, University of Colorado Denver, Aurora, USA
               type:PostalAddress
            type:Organization
      name:Enos Kline
      affiliation:
            name:University of Colorado Denver
            address:
               name:Department of Medicine, University of Colorado Denver, Aurora, USA
               type:PostalAddress
            type:Organization
            name:University of Washington
            address:
               name:Present Address: Department of Bioengineering, University of Washington, Seattle, USA
               type:PostalAddress
            type:Organization
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      affiliation:
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            address:
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               type:PostalAddress
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      name:Department of Medicine, University of Colorado Denver, Aurora, USA
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      name:Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, USA
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