GSM6837008: 86-12-PanCK+; Homo sapiens; RNA-Seq
All human tissue was sourced from the Skin Cancer Prevention Program Biorepository at the University of Arizona Cancer Center. The protocol has been approved by the Institutional Review Board at the University of Arizona (Protocol Title: Biomarker development for early phase melanoma prevention trials; Protocol Number: 2012277005). A set of matched sun protected and actinic keratosis from two individuals were used for in this study. Tissue blocks were sectioned with a 5 µm thickness and mounted in the center of the slide (Superfrost Plus Micro slides, VWR) within an area of 35.3 mm long by 14.1 mm wide to allow detection by the NanoString GeoMx DSP instrument. Sample preparation was performed following Leica Biosystems Bond RX FFPE RNA Slide Preparation Protocol in the GeoMx NGS Slide Preparation User Manual (NanoString, MAN-10115-03 for software version v2.1). This assay relies upon photocleavable RNA probes coupled to oligonucleotide tags that allow the selection of tissue compartments of interest for RNA analysis. Slides were baked at 60°C for 60 minutes. Samples were loaded into Leica Bond Rx for deparaffinization and antigen retrieval with the following settings; Bake and Dewax preparation program, 150µl buffer dispense volume, HIER 20 minutes with ER2 at 100°C, 1µ/mL proteinase K for 15 minutes. Slides were briefly stored in 1x PBS. Excess PBS was removed from slides before the addition of 200µL Whole Transcriptome Assay (WTA) probe sets diluted 1:10 in Buffer R (NanoString), covered with Hybrislips (Grace Biolabs, 714022) and incubated at 37°C overnight in a hybridization chamber lined with Kimwipes moistened with 2x SSC. Unbound probes were removed by washing twice at 37°C for 25 minutes in 50% Formamide (ThermoFisher, AM9324)/2x SSC followed by two washes at room temperature in 2x SSC for 2 minutes. Slides were blocked with 200µL Buffer W (NanoString) for 30 minutes followed by 1 hour incubation at room temperature in a humidity chamber with morphology marker solution comprised of 1µL PMEL17 (Novus Biologicals, NBP2-34638AF647), 2µL CD45 (Cell Signaling Technology, 13917BF), 0.66µL Pan-cytokeratin (Novus Biologicals, NBP2-33200AF488), 10µL Syto83 (ThermoFisher, S11364) in Buffer W (NanoString) to 200µL per slide. Slides were subsequently washed twice with 2x SSC before loading onto NanoString GeoMx DSP instrument.  Slides were loaded and scanned on GeoMx instrument according to GeoMx DSP Instrument User Manual (NanoString, MAN-10116-03 for software version v2.1) with the following scan exposure times; 200ms FITC/525nm, 60ms Cy3/568nm, 250ms Texas Red/615nm, 300ms Cy5/666nm. 12 polygonal regions of interest (ROI) were selected per slide with segmentation masks applied to each ROI selecting Pan-cytokeratin positive and Pan-cytokeratin negative areas within each ROI. Channel thresholds were adjusted to maximize inclusion of signal areas and minimize inclusion of non-signal areas with default settings for erode, N-dilate, hole size and particle size