AbstractNeuroscience research greatly benefits from single‐cell sequencing technologies, which can reveal transcriptional alterations on a cellular level. However, preparing single‐cell suspensions is technically challenging, requires experience, and has several limitations that can influence the transcriptional readout. Performing sequencing of single nuclei instead of single cells alleviates several of the challenges of sample preparation and highlights acute nuclear transcription. Here, we provide a protocol to prepare a nuclei suspension for single‐nucleus RNA‐sequencing for cell type‐specific transcriptional profiling of brain tissue using the 10x Genomics single‐cell gene expression assay. Furthermore, we highlight important aspects to consider during experimental design and data analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Preparation of single‐nucleus suspensionBasic Protocol 2: Preparation and sequencing of single‐nucleus libraries for RNA‐seq