Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. 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 artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating miRNA-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. Overall design: Argonaute HITS-CLIP on the MCF-7 breast cancer cell line treated with 17ß-estradiol for 0, 6 or 24 hours using a nested reverse transcriptions pimer and protected or unprotected reverse PCR primers for library amplification.
External Link: /pubmed:27150721