GSM9371931: Pt3-2; Homo sapiens; OTHER
For each tissue section, eight circular regions of interest (ROIs; 300 μm diameter) were selected. Within each ROI, three biological segments—immune, tumor, and stromal—were independently profiled FFPE tissue sections were processed using the NanoString GeoMx Digital Spatial Profiling (DSP) protocol with the Cancer Transcriptome Atlas (CTA) RNA probe panel. Slides were deparaffinized, subjected to antigen retrieval, and incubated with CTA RNA probe sets together with fluorescent morphology markers to enable ROI selection. After overnight hybridization, regions of interest (ROIs) were selected based on immunofluorescent segmentation markers, and oligonucleotide tags linked to the CTA probes were released by UV photocleavage. Photocleaved indexing oligos from each ROI were collected into separate wells and amplified by PCR using Illumina-compatible adapters and ROI-specific indices. Amplified libraries were purified, quantified, and pooled to create the final sequencing library. Sequencing was performed on an Illumina instrument following NanoString recommendations for CTA panel libraries.