Determining mRNA decay rates using RNA approach to equilibrium sequencing (RATE-seq)

Farah Abdul-Rahman, David Gresham

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages15-24
Number of pages10
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1720
ISSN (Print)1064-3745

Keywords

  • 4-thiouracil
  • Metabolic labeling
  • RNA degradation
  • RNA stability
  • RNA turnover

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Abdul-Rahman, F., & Gresham, D. (2018). Determining mRNA decay rates using RNA approach to equilibrium sequencing (RATE-seq). In Methods in Molecular Biology (pp. 15-24). (Methods in Molecular Biology; Vol. 1720). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7540-2_2