Polysome profiling combined with RNA sequencing provides a validated protocol to quantify translationally silent ribosome induction in yeast and mammalian cells.
This paper provides a detailed experimental protocol for measuring translationally silent ribosomes in eukaryotic cells.
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Translationally silent ribosomes lack bound mRNA and are difficult to quantify. Here, we present a protocol to measure silent ribosome induction under diverse conditions in yeast and mammalian cells. We describe steps to isolate polysome-profiling fractions, analyze ribosome-associated RNA by RNA sequencing with identification and removal of anomalously amplified rRNAs, and validate measurements by qPCR. For complete details on the use and execution of this protocol, please refer to Rahaman et al. 1 • Steps for detecting silent ribosomes in eukaryotic cells • Guidance on polysome profiling and spike-in RNA normalization • Instructions for correcting anomalous 5S rRNA amplification • RNA-seq dataset from heat-stressed macrophages Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Translationally silent ribosomes lack bound mRNA and are difficult to quantify. Here, we present a protocol to measure silent ribosome induction under diverse conditions in yeast and mammalian cells. We describe steps to isolate polysome-profiling fractions, analyze ribosome-associated RNA by RNA sequencing with identification and removal of anomalously amplified rRNAs, and validate measurements by qPCR.
Rahaman et al. (Fri,) reported a other. Polysome profiling combined with RNA sequencing provides a validated protocol to quantify translationally silent ribosome induction in yeast and mammalian cells.
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