Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation.

Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used.

Image-Based Screening for Stress Granule Regulators.

Stress granule (SG)-based RNA interference (RNAi) screening is a powerful method to discover factors that control protein synthesis and aggregation, as well as regulators of SG assembly and disassembly. Here, we describe how to set up and optimize a large-scale siRNA screen, and give a detailed outline for the automated quantification of SGs as a visual readout. Hit evaluation via calculated Z scores provides a list of candidates for further in-depth studies.