|
| Status |
Public on Feb 19, 2016 |
| Title |
Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts. |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
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.
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| Overall design |
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.
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| |
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| Contributor(s) |
Gillen AE, Yamamoto TM, Kline E, Hesselberth JR, Kabos P |
| Citation(s) |
27150721 |
| |
| Submission date |
Feb 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Kabos |
| Organization name |
University of Colorado School of Medicine
|
| Department |
Division of Medical Oncology
|
| Street address |
12801 E. 17th Avenue
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
|
| Samples (6)
|
| GSM2065792 |
MCF7+E2 0h AGO HITS-CLIP (Nested, Protected) |
| GSM2065793 |
MCF7+E2 6h AGO HITS-CLIP (Nested, Protected) |
| GSM2065794 |
MCF7+E2 24h AGO HITS-CLIP (Nested, Protected) |
|
| Relations |
| BioProject |
PRJNA312501 |
| SRA |
SRP070520 |
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