This unit describes a protocol for genome-wide identification of translationally regulated genes during embryonic stem cell differentiation using integrated transcriptome and translation state profiling. Actively translated mRNAs associated with multiple ribosomes (known as polysomes) and translationally inactive mRNAs sequestered in messenger ribonucleoprotein particles (mRNPs), can be separated by sucrose gradient fractionation based on size. Because the number of ribosomes on a transcript correlates with the rate of synthesis of its encoded protein, this allows an operational distinction between well-translated and poorly translated mRNA molecules. In this analysis, fractionated mRNA and total RNA are used to probe microarrays to identify differentially translated genes.