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bioRxiv · 10.64898/2026.04.03.716374

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

George, B.·Kirkpatrick, B. Q.·Zhang, Q.
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Abstract

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/716374v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f75556org.highwire.dtl.DTLVardef@103bb8eorg.highwire.dtl.DTLVardef@e7a9eorg.highwire.dtl.DTLVardef@942768_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBenchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brain C_LIO_LIScales across tissue inputs (e.g., hippocampus [~]15-20 mg; cerebellum [~]50-70 mg) without ultracentrifugation or 15 mL gradients C_LIO_LIMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows. C_LI

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George, B., Kirkpatrick, B. Q., Zhang, Q.. 2026-04-07. Myelin-Free Nuclei Isolation from Mouse Hippocampus and Cerebellum for snRNA-Seq with Benchtop Gradient Centrifugation. https://doi.org/10.64898/2026.04.03.716374

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