This protocol presents a plate-based workflow to perform RNA sequencing analysis of single cells/nuclei using Smart-seq2. We describe (1) the dissociation procedures for cell/nucleus isolation from the mouse brain and human organoids, (2) the flow sorting of single cells/nuclei into 384-well plates, and (3) the preparation of libraries following miniaturization of the Smart-seq2 protocol using a liquid-handling robot. This pipeline allows for the reliable, high-throughput, and cost-effective preparation of mouse and human samples for full-length deep single-cell/nucleus RNA sequencing. For complete details on the use and execution of this protocol, please refer to Bowers et al. (2020).