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Series GSE68471 Query DataSets for GSE68471
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
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.
Overall design Libraries were prepared essentially as described in Slevin et al. (Mol. Cell 53: 1020-1030, 2014)
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
Organization name The University of North Carolina at Chapel Hill
Street address 207 Fordham Hall
City Chapel Hill
State/province NC
ZIP/Postal code 27599
Country USA
Platforms (2)
GPL15520 Illumina MiSeq (Homo sapiens)
GPL16479 Illumina MiSeq (Drosophila melanogaster)
Samples (5)
GSM1673355 Run1_Combined_data_5_files
GSM1673356 Run2_Combined_data_5_files
GSM1673357 DM_CTTGT_L001_R1_001
BioProject PRJNA282807
SRA SRP057878

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Supplementary file Size Download File type/resource
GSE68471_RAW.tar 98.8 Mb (http)(custom) TAR (of TXT)
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Raw data are available in SRA
Processed data provided as supplementary file

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