Protocol to analyze steps of ribosome biogenesis in nuclear and cytosolic fractions of yeast cells

Cell fractionation enables the study of compartment-specific RNA processing events in organisms with complex cell walls. Here, we present a protocol for isolating nuclear and cytosolic fractions from Saccharomyces cerevisiae. We describe spheroplast formation using zymolyase, followed by subcellular fractionation via a sucrose density gradient. Finally, we detail the extraction of high-quality RNA from these fractions. This approach overcomes cell wall barriers to allow the analysis of ribosome biogenesis. For complete details on the use and execution of this protocol, please refer to Zadali et al.¹.