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| Status |
Public on Dec 31, 2019 |
| Title |
Chemical nucleoside conversion-based measurement of RNA stability by targeted and untargeted mRNA 3ยด end sequencing |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Gene expression profiling by high-throughput sequencing determines changes in gene expression only at steady state but prevents our understanding of the underlying gene expression kinetics. Here, we describe a protocol that combines metabolic RNA labeling with thiol-specific chemical nucleoside conversion to determine the stability of polyadenylated RNA transcripts. And we provide a targeted mRNA 3 end library preparation protocol that enable to robustly determine the stability even of RNA transcripts that escape robust detection in untargeted libraries. The described methods enable cost-effective insights into the kinetics underlying steady-state gene expression in order to study the mechanisms underlying the regulation of gene expression at a transcript-specific and genomic scale.
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| Overall design |
Mouse embryonic stem cells (mES cells) were subjected to s4U metabolic RNA labeling for 12h, followed by washout and chase using non-thiol-containing uridine. Total RNA was prepared at various time points along the chase (0h, 0.5h, 1h, 3h, 6h, and 12h). Total RNA was then subjected to alkylation and targeted RNA-seq using custom-designed primers.
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| Contributor(s) |
Herzog VA, Fasching N, Ameres SL |
| Citation missing |
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| |
| Submission date |
Aug 03, 2018 |
| Last update date |
Jan 01, 2020 |
| Contact name |
Brian Reichholf |
| E-mail(s) |
brian.reichholf@imba.oeaw.ac.at
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| Organization name |
IMBA
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| Lab |
Stefan Ameres
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| Street address |
Doktor-Bohr-Gasse 3
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platforms (1) |
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| Samples (14)
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| Relations |
| BioProject |
PRJNA484507 |
| SRA |
SRP156338 |
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