title: Spatial transcriptomic profiling of primary and recurrent RCAS-derived glioblastoma in a mouse model using 10x Genomics Visium v1
library_construction_protocol: Fresh tumors and healthy brain tissue were dissected from Nestin-Tv-a RCAS mice. Animals were sacrificed according to institutional ethical guidelines, and brains were rapidly dissected. Tumor and control brain regions were embedded in OCT on dry ice and snap-frozen in isopentane. Samples were stored at –80 °C until cryosectioning.  For RNA quality control, 10 × 5 µm tissue sections from each tumor block were collected into microtubes and processed with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration and integrity were assessed on an Agilent Bioanalyzer. Only samples with RIN > 8.8 were advanced to Visium library preparation.  Frozen tissue was cryosectioned at 10 µm on a pre-cooled cryostat and mounted onto 10x Genomics Visium Spatial Gene Expression Slides. Sections were fixed in ice-cold methanol, stained with hematoxylin and eosin (H&E), and imaged using a 10× objective including the fiducial frame. Permeabilization was performed for 21 minutes, based on prior optimization for this tissue type. Subsequent steps — second-strand synthesis, cDNA amplification, and library construction — were performed using the Visium Spatial Gene Expression Reagent Kit (10x Genomics, CG000239 Rev G) according to the manufacturer's protocol.