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
Bibliographical noteFunding 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 ).
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- Cell Biology
- Cell Differentiation
- Molecular Biology
- Stem Cells