Protocol for Quantifying Silent Ribosome Induction using Polysome Profiling

Translationally silent ribosomes have become an important focus in RNA biology, yet their quantification remains challenging. Lacking mRNAs, they represent a hibernating ribosome state. This protocol outlines how to measure silent ribosome induction under diverse conditions by analyzing RNA from polysome-profiling fractions in yeast and mammalian cells, using RNA-seq with removal of anomalously amplified rRNAs and qPCR for validation. Overall design: For an example of the execution of this protocol, please refer to Rahaman, et. al.1