Protocol to characterize tumor microenvironment in a diffuse midline glioma mouse model using multi-omics approach on sequential sections.

Diffuse midline glioma (DMG) tumors form complex tumor-immune microenvironments with marked cellular heterogeneity. Here, we present a protocol to generate orthotopic DMG tumors in immunocompetent neonatal mice and to spatially characterize the tumor ecosystem using a multi-omics workflow on sequential brain sections. We detail procedures for brain freezing, cryosectioning, single-nuclei RNA sequencing, and Visium spatial transcriptomics. We then describe steps for cyclic immunofluorescence and integrative computational analysis to resolve cellular states and spatial organization. For complete details on the use and execution of this protocol, please refer to Collot et al.1.