title: Spatial transcriptomic profiling of healthy brain and RCAS-derived glioblastoma in a mouse model using 10x Genomics Visium HD
library_construction_protocol: Mouse brain tissue (one healthy and one RCAS-derived glioma) was collected and post-fixed in 4% paraformaldehyde for 24 hours, dehydrated, and embedded in paraffin. Sections of 3 µm thickness were cut from the FFPE blocks and mounted onto standard glass slides for histological preparation and subsequent spatial transcriptomic processing.  RNA quality was assessed from each FFPE block by processing ten 5 µm sections using the RNeasy FFPE Kit (Qiagen 73504). Extracted RNA was analyzed on an Agilent Bioanalyzer to determine DV200 values and confirm that they met the recommended threshold for Visium HD spatial transcriptomics.  Tissue sections mounted on glass slides were H&E stained prior to hybridization. Probe hybridization was performed using the Visium Mouse Transcriptome Probe Set v2.0 (mm10-2020-A), supplemented with custom-designed probes targeting RCAS-derived transgenes (one probe for human PDGFB and two probes for RFP). Following hybridization, slides were processed using the Visium CytAssist instrument to transfer probe-labeled tissue onto Visium HD slides.