Protocol for isolating myelin-reduced nuclei from mouse hippocampus and cerebellum for snRNA-seq using benchtop gradient centrifugation

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing (snRNA-seq) because myelin and debris can reduce sample quality. Here, we present a protocol for producing intact, debris-reduced nuclei from mouse hippocampus and cerebellum for snRNA-seq. We describe controlled mechanical homogenization, low-volume sucrose-gradient pelleting using a standard benchtop centrifuge, repeated nuclei resuspension buffer (NSB) washes, and optional magnetic enrichment for debris-rich preparations. This protocol supports downstream 10× Genomics Flex and PARSE WT library preparation.