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| Status |
Public on Jun 01, 2015 |
| Title |
EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates |
| Organisms |
Drosophila melanogaster; Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3’ ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3’ ends contain posttranscriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3’ end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3’ ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nts at the 3’ end of mature histone mRNA maintaining the length of the histone transcripts. Histone genes in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3’ additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.
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| Overall design |
Libraries were prepared essentially as described in Slevin et al. (Mol. Cell 53: 1020-1030, 2014)
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| Contributor(s) |
Marzluff WF, Welch JD |
| Citation(s) |
26015596, 30408609 |
| Submission date |
May 01, 2015 |
| Last update date |
Jun 01, 2020 |
| Contact name |
William F Marzluff |
| E-mail(s) |
marzluff@med.unc.edu
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| Organization name |
The University of North Carolina at Chapel Hill
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| Street address |
207 Fordham Hall
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platforms (2) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL16479 |
Illumina MiSeq (Drosophila melanogaster) |
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| Samples (5)
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| Relations |
| BioProject |
PRJNA282807 |
| SRA |
SRP057878 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE68471_RAW.tar |
98.8 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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