Calculating RNA degradation rates using large-scale normalization in mouse embryonic stem cells

Juliane Oliveira Viegas; Lior Fishman; Eran Meshorer; Michal Rabani

doi:10.1016/j.xpro.2023.102534

Calculating RNA degradation rates using large-scale normalization in mouse embryonic stem cells

STAR Protoc. 2023 Sep 15;4(3):102534. doi: 10.1016/j.xpro.2023.102534. Epub 2023 Sep 5.

Authors

Juliane Oliveira Viegas¹, Lior Fishman¹, Eran Meshorer², Michal Rabani³

Affiliations

¹ Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 9190401, Israel.
² Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 9190401, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 9190401, Israel. Electronic address: eran.meshorer@mail.huji.ac.il.
³ Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 9190401, Israel. Electronic address: michal.rabani@mail.huji.ac.il.

Abstract

Data normalization is critical to the process of estimating RNA degradation by analyzing RNA levels when transcription is blocked. Here, we present a protocol for measuring mRNA degradation rates, optimized for mouse embryonic stem cells, using α-amanitin inhibitor. We describe steps for a time course α-amanitin treatment, RNA-seq, and alignment; we then detail procedures for analyzing data and sequence enrichment. Our method relies on large-scale normalization of stable transcripts in genomic RNA-seq measurements, providing reliable readouts. For complete details on the use and execution of this protocol, please refer to Viegas et al.¹.

Keywords: Cell Biology; Cell Differentiation; Molecular Biology; RNA-seq; Stem Cells.

MeSH terms

Alpha-Amanitin* / pharmacology
Animals
Genomics
Mice
Mouse Embryonic Stem Cells*
RNA / genetics
RNA-Seq

Substances

Alpha-Amanitin
RNA