Transcriptomics

Dataset Information

298

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. 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. Argonaute HITS-CLIP on the MCF-7 breast cancer cell line treated with 17β-estradiol for 0, 6 or 24 hours using a nested reverse transcriptions pimer and protected or unprotected reverse PCR primers for library amplification.

ORGANISM(S): Homo sapiens  

SUBMITTER: Tomomi M Yamamoto   Jay R Hesselberth  Peter Kabos  Enos Kline  Austin E Gillen 

PROVIDER: E-GEOD-78059 | ArrayExpress | 2016-02-19

SECONDARY ACCESSION(S): SRP070520GSE78059PRJNA312501

REPOSITORIES: GEO, ArrayExpress, ENA

Similar Datasets

2014-08-04 | E-GEOD-57855 | ArrayExpress
| GSE110260 | GEO
2012-10-21 | E-GEOD-40778 | ArrayExpress
| PRJNA257772 | ENA
| PRJNA268387 | ENA
| PRJNA214606 | ENA
| PRJNA155915 | ENA
2015-05-08 | E-GEOD-63604 | ArrayExpress
2015-05-08 | E-GEOD-63597 | ArrayExpress
2016-02-17 | E-GEOD-53696 | ArrayExpress