A translating ribosome protects a discrete fragment of ∼30 nt on its mRNA template from nuclease digestion [Steitz, JA, (1969) Nature 224, 957]. Ribosome profiling determines the positions of active ribosomes on cellular mRNA by deep sequencing the ribosome-protected mRNA fragments [Ingolia, NT et al. (2009) Science 324, 218]. Quantitative measurement of abundance in this complex library is obtained by counting the number of times any given sequence is read by deep sequencing. The rate of protein synthesis is measured by examining the ribosome density of each mRNA.Current methods to isolate polysomes and monosomes rely on several hours of ultracentrifugation using either a sucrose gradient or a cushion. We have investigated size-exclusion chromatography (SEC) in disposable spin-columns as an alternative to ultracentrifugation to isolate polysomes and monosomes for ribosome profiling. The size-exclusion method is simpler and rapid, and does not require any special equipment. Using this method we have streamlined the steps to convert ribosome-protected RNA fragments into libraries compatible with sequencing on Illumina® instruments.We present ribosome profiling data generated using these methods in conjunction with bioinformatics tools for analyzing the deep sequencing data in mammalian samples.
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