2158_010_Segmented_Epithelioid_p
Spatial Transcriptomic was performed by GeoMx DSP® (Nanostring technologies)starting from slides of 5μm FFPE tumor tissue.  In order to perform spatial selection, slides were stained with GeoMx Solid Tumor TME Morphology Kit including antibodies against CytoKeratin (CK) and nuclear stain SYTO-13.  For each slide, area of interest (AOI) were selected by two pathologists as representative of the tumor heterogeneity, based on morphology and expression of the Pan-CK marker. Three types of regions were selected: area including only epithelioid cells, area including only sarcomatoid cells and transitional regions containing both mixed epithelioid and sarcomatoids cells. These transitional regions were further segmented to separate the Epithelioid and sarcomatoid component.  The slides were hybridized with the GeoMx® Cancer Transcriptome Atlas panel according to manufacturer protocol. After AOIs selection and photo-cleavage GeoMx RNA probes were sequenced. Hybridized RNA-probes were collected from each of the selected AOIs, and libraries for NGS sequencing were constructed by GeoMx® Seq Code Pack (Nanostring technologies). Libraries were pooled according to AOIs dimension, purified by AMPure XP beads (Beckman Coulter, Brea, Cali-fornia, USA) clean up and finally resuspended in a volume proportional to the number of pooled AOIs. The quality and quantity of library pools were evaluated by Agilent Bioanalyzer.