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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• RNA-seq
• Molecular Biology
• Stem Cells
• Cell Differentiation