title: GSM8439734: 39 PV/N 22000481 stroma; Mus musculus; OTHER
library_construction_protocol: Slides were processed within 5 days after the microtomy for the NanoString GeoMx DSP RNA assay following the GeoMx NGS Slide Preparation User Manual (NanoString, MAN-10 115-04). After 30 min baking at 60oC, dewaxing, heat-induced epitope retrieval (ER2 for 20 min at 100oC) and proteinase K treatment (1 mg/mL proteinase K in 1X phosphate-buffered saline (PBS) at 37°C for 15 min) was performed on Leica Biosystems BOND RX according to the FFPE RNA Slide Preparation Protocol. Slides were hybridized with the GeoMx Whole Transcriptome Atlas Mouse RNA for Illumina Panel (GMX[1]RNA-NGSMsWTA) overnight at 37oC, followed by a series of stringent washes and morphology staining: Syto 13 for nuclei, pancytokeratin- AF532 to visualize epithelial cells and smooth muscle actin (aSMA-AF647) to visualize stroma. The staining was visualized on the GeoMx DSP instrument using a 20X objective on FITC, CY3, Texas Red and CY5 channels. A total of 190 regions of interest (ROI) were selected from 13 mouse uterus sections (6 Thra1 PV/+ and 7 wild-type mice), which included endometrial mucosa, glands, and stroma; a minimum of 5 ROIs were taken per category per mouse. Annotations were selected by a board-certified veterinary pathologist based on morphology and marker staining using HALO (Indica Labs). ROI masks were imported to the GeoMx and used to guide UV light cleavage of the barcode linkers of the prebound RNA probes in each area of illumination, and the cleaved probes were deposited in DSP collection plates. Library construction and sequencing (NextSeq2000) was performed according to Nanostring's Guidelines at the Genomics Core (OSTR, CCR). Processed data was uploaded to the DSP analysis suite for visualization and quality control while detailed data analysis was performed using an R-package from Nanostring.