Please note that we have stopped the regular imports of Gene Expression Omnibus (GEO) data into ArrayExpress. This may not be the latest version of this experiment.

E-GEOD-78059 - Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts

Status
Released on 19 February 2016, last updated on 27 February 2016
Organism
Homo sapiens
Samples (6)
Protocols (3)
Description
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.
Experiment type
other 
Contacts
Peter Kabos <geo@ncbi.nlm.nih.gov>, Austin E Gillen, Enos Kline, Jay R Hesselberth, Tomomi M Yamamoto
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-GEOD-78059.idf.txt
Sample and data relationshipE-GEOD-78059.sdrf.txt
Processed data (1)E-GEOD-78059.processed.1.zip
Links