Protocol for mechanical nuclei dissociation and isolation from frozen tissue and organoids for single-nucleus RNA sequencing.

Preserving RNA integrity in complex tissues such as the brain is challenging. Here, we present a protocol for isolating intact nuclei from frozen tissue and organoids for single-nucleus RNA sequencing (snRNA-seq). We describe steps for mechanical dissociation, using the GentleMACS Dissociator, serial filtration, and magnetic bead-based nuclei isolation. We then detail nuclei quality control and counting. This approach yields pure single-nucleus suspensions suitable for downstream fixation, cryopreservation, or library preparation across diverse tissue types. For complete details on the use and execution of this protocol, please refer to Guillon et al.1.