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

Juliane Oliveira Viegas, Lior Fishman, Eran Meshorer^*, Michal Rabani^*

^*Corresponding author for this work

Department of Genetics

Research output: Contribution to journal › Article › peer-review

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

Original language	American English
Article number	102534
Journal	STAR Protocols
Volume	4
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2023.102534
State	Published - 15 Sep 2023

Bibliographical note

Funding Information:
This work was supported by the Israel Ministry of Science ( MOST 0004272 to E.M.), the European Innovation Council (EIC) Horizon Europe ( RT-SuperES 101099654 to E.M.), and the European Research Council (ERC) Horizon 2020 grant ( 852451 to M.R.). E.M. is the incumbent of the Arthur Gutterman Professor Chair for Stem Cell Research. J.V. is supported by the European Union’s Horizon -2020 Marie Skłodowska Curie EpiSyStem ITN Network ( 765966 ).

Publisher Copyright:
© 2023 The Authors

Keywords

Cell Biology
Cell Differentiation
Molecular Biology
RNA-seq
Stem Cells

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10.1016/j.xpro.2023.102534

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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",

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AU - Rabani, Michal

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AB - 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

KW - Cell Biology

KW - Cell Differentiation

KW - Molecular Biology

KW - RNA-seq

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

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