GSM8853694: Dog_7_deep_3; Canis lupus familiaris; OTHER
GeoMx DSP analysis was performed according to NanoString's standard procedures for NGS readout. Briefly, semi-automated slide preparation was conducted on a Leica BOND RX stainer for deparaffinization, epitope retrieval, and proteinase K treatment to expose RNA targets. The GeoMx Canine Cancer Atlas probe set was hybridized in situ to the tissue sections by overnight incubation at 37C. Subsequently, tissue sections were stained with fluor-conjugated morphology marker antibodies specific for pan-cytokeratin (PanCK; Alexa 532, Cy3/568nm channel), CD45 (Alexa 594, Texas Red/615nm channel), CD3 (Alexa 647,  Cy5/666 nm channel), and the SYTO-13 nucleic acid stain (FITC/525nm channel, for instrument focus).  Morphology markers facilitated selection of regions of interest (ROIs) containing approximately 300 cells, as defined by the nuclear stain. Four ROIs were selected for each phenotype (5 phenotypes: surface normal, surface tumor, surface peri-tumoral dysplasia, deep invasive tumor, tumor microenvironment), leading to a total of approximately 20 ROIs per patient. indexed libraries were prepared from the DSP DNA oligonucleotides using high-fidelity PCR with GeoMx Seq Code Illumina-compatible primers. Validated libraries (i.e., expected amplicon size of 162 bp) were sequenced on an Illumina NovaSeq X Plus Sequencing System