Myelin-Free Nuclei Isolation from Mouse Hippocampus and Cerebellum for snRNA-Seq with Benchtop Gradient Centrifugation

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing because myelin and debris can reduce nuclei quality. We describe an optimized protocol for mouse hippocampi and cerebella using tube-and-pestle homogenization, low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, and magnetic enrichment as the recommended final cleanup step to reduce debris/non-nuclear carryover. Under the tested conditions, the workflow produces intact, debris-reduced nuclei and supports downstream 10x Genomics Flex and PARSE WT library preparation.