Simple SummaryGene expression studies provide valuable insights into the mechanisms underlying biological processes, including aging. RNA sequencing can be used to identify changes in gene expression across the entire genome. While normally, RNA sequencing is performed on multiple biological replicates that are sequenced individually, the results of this study show that similar results can be obtained by pooling those individual samples together before sequencing. Pooling RNA samples prior to sequencing will reduce the cost of experiments, which may allow for additional investigations and provide more information about the mechanisms involved.AbstractAnalysis of gene expression changes across the genome provides a powerful, unbiased tool for gaining insight into molecular mechanisms. We have effectively used RNA sequencing to identify differentially expressed genes in long-lived genetic mutants inC. elegansto advance our understanding of the genetic pathways that control longevity. Although RNA sequencing costs have come down, cost remains a barrier to examining multiple strains and time points with a sufficient number of biological replicates. To circumvent this, we have examined the efficacy of identifying differentially expressed genes by sequencing a pooled RNA sample from long-livedisp-1mitochondrial mutant worms. We found that sequencing a pooled RNA sample could effectively identify genes that were found to be significantly upregulated in the two individually sequenced RNA-seq experiments. Finally, we compared the genes significantly upregulated in the two individually sequenced RNA-seq experiments to two previous microarray experiments to come up with a high-confidence list of modulated genes in long-livedisp-1mutant worms. Overall, this work demonstrates that RNA sequencing of pooled RNA samples can be used to identify differentially expressed genes.