| Chapter title |
Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq)
|
|---|---|
| Chapter number | 2 |
| Book title |
Methods in Molecular Biology
|
| Published in |
Methods in molecular biology, December 2017
|
| DOI | 10.1007/978-1-4939-7540-2_2 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7539-6, 978-1-4939-7540-2
|
| Authors |
Abdul-Rahman, Farah, Gresham, David, Farah Abdul-Rahman, David Gresham |
| Abstract |
RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment. The analysis of the RATE-seq data entails normalization of RNAseq libraries to thiolated RNA spike-ins and nonlinear model fitting to estimate the degradation rate constant for each RNA species. |
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