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

UNLABELLED: 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 and low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, with optional magnetic enrichment of nuclei 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.

HIGHLIGHTS: Benchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brainScales across tissue inputs (e.g., hippocampus ∼15-20 mg; cerebellum ∼50-70 mg) without ultracentrifugation or 15 mL gradientsMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows.