Optimized nuclei isolation and snRNA-seq reveal oligodendrocyte pathway dysregulation in MOGHE brain tissue from pediatric patients.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for exploring cellular diversity, but isolating intact cells from complex tissues like the brain remains challenging. Single-nucleus RNA sequencing (snRNA-seq) overcomes these limitations by profiling nuclear RNA from frozen or archived tissue, reducing dissociation bias. Here, we present a simplified protocol for nuclei isolation from frozen human brain biopsies optimized for high yield and minimal debris, enabling robust snRNA-seq analysis. To validate it, we applied the protocol to biopsies from two pediatric patients with mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE) carrying somatic SLC35A2 variants. snRNA-seq of isolated nuclei revealed a marked increase in oligodendrocytes in MOGHE samples, consistent with histopathological observations. Differential gene-expression analysis of oligodendrocyte-derived nuclei showed potential dysregulation of key pathways, including NOTCH, WNT/β-catenin, SLIT/ROBO, and Rho-GTPases signaling, as well as pathways associated with oxidative stress and neuroinflammation, and impaired neuron-glia communication. Despite advances in transcriptomic chemistries allowing fixed-cell analyses, reliable and debris-free nuclei isolation remains essential for generating high-quality single-nucleus data. Our streamlined protocol offers a reproducible, adaptable approach compatible with current and emerging snRNA-seq technologies.