Protocol for determining the contribution of protein and RNA to condensate organization by permeabilizing and enzyme-treating live U-2 OS cells.

Ribonucleoprotein granules are comprised of protein and RNA. We present a protocol for querying the relative contribution of protein and RNA interactions to granule organization by permeabilizing and treating cells with proteinase or RNase enzymes. We then detail steps for performing single-molecule fluorescence in situ hybridization (smFISH) on enzyme-treated samples for RNA visualization. Finally, we describe detailed instructions for quantifying results generated with this protocol. This protocol can potentially query the contribution of protein-RNA, protein-protein, and RNA-RNA interactions to the organization of any intracellular granule. For complete details on the use and execution of this protocol, refer to Parker et al.(1).