Eliminating Ultracentrifugation in the Deep Sequencing of Ribosome-Protected mRNA Fragments Using Polysomes and Monosomes.

RNA abundance measured either by microarrays or high-throughput sequencing reflects protein levels for transcripts that are not subjected to translation control. Isolation of translating ribosomes (polysomes and monosomes) and analysis of the associated mRNAs provide a better measure of translation rates to estimate the copies of the synthesized protein. Current methods to isolate polysomes and monosomes rely on ultracentrifugation, using either a sucrose gradient or a cushion. While the sucrose cushion avoids the need for a more specialized gradient fractionation step, it still requires access to an ultracentrifuge and several hours of centrifugation. We have investigated size-exclusion chromatography as an alternative to ultracentrifugation for isolating polysomes. As reported in the literature, we found polysomes in the void volume of commonly used size-exclusion resins. The size-exclusion method is simpler, rapid, and does not require any special equipment. We describe the use of size-excluded polysomes for ribosome profiling using the method of Ingolia et al. (Science 324, 218-223 [2009]) to monitor protein production in cells and define the proteome.