Glioblastoma (GBM) is the most common and lethal primary adult brain tumor. It represents a rapidly evolving ecosystem, including heterogeneous tumor cells and an immunosuppressive tumor microenvironment (TME) with diverse non-malignant cells. High-throughput single-cell omics profiling, such as single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq, respectively), is an emerging powerful tool to deconvolute diverse cell types and states as well as intricate cellular and molecular functions and interactions that underlie the complex GBM ecosystem. snRNA-seq is a methodology that profiles the transcriptome using isolated nuclei instead of intact cells. It is an alternative to scRNA-seq and is compatible with frozen samples and difficult-to-dissociate tissues, such as brain or brain tumor tissues. However, efficient, optimized procedures are instrumental in preparing high-quality single nuclei from clinical GBM specimens and patient-derived GBM cell lines across different conditions for snRNA-seq. Here, we provide a toolkit of detailed protocols for nucleus isolation, counting, and quality control, enabling a streamlined single-nucleus preparation for snRNA-seq characterization.