Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries. This protocol can be adapted for transcriptome analysis of oocytes from other species and be used to generate high-quality data from single embryos. For complete details on the use and execution of this protocol, please refer to Biase and Kimble (2018).