Stress granules are mRNA-protein assemblies formed on nontranslating mRNAs. Stress granules are important in the stress response, related to neuronal mRNP granules, and aberrant stress granules contribute to some degenerative diseases. By RNA-Seq and single molecule FISH, we describe the stress granule transcriptome in both yeast and mammalian cells. This reveals that while essentially every mRNA, and some ncRNAs, can be targeted to stress granules, the efficiency of targeting can vary from <1% to 73%. mRNA accumulation in stress granules is increased by longer coding regions, poor translatability, and correlates with some RNA binding proteins. Standardizing the RNA-Seq analysis by single molecule FISH allows a quantitative description of the general and stress gra­nule transcriptome. Approximately 15% of the bulk mRNA molecules accumulate in stress granules suggesting their effect will be limited primarily to subsets of mRNAs highly accumulating in stress granules Overall design: To determine the RNAs in mammalian stress granules, we purified and performed RNA-Seq in triplicate on SG cores from U-2 OS cells isolated following 60’ of arsenite exposure, referred to as SGRNA. For each sample, 5% of the lysate was extracted for RNA-Seq on the total RNA population, referred to as TotalRNA. To determine the RNAs in yeast stress granules, we purified and sequenced the mRNAs within SG cores from Saccharomyces cerevisiae after induction of SGs by sodium azidefor 30 minutes. SGRNA and TotalRNA were isolated from three independent biological replicates and sequenced.