title: GSM9679412: Slide 5; ROI 011; Segment: CD45; Homo sapiens; OTHER
library_construction_protocol: Formalin-fixed, paraffin-embedded (FFPE) breast tissues from patients diagnosed with ductal carcinoma in situ (DCIS) or invasive ductal carcinoma (IDC) were obtained from the Department of Pathology at the University of Oklahoma Health Campus (Oklahoma City, OK). A pathologist confirmed the histopathologic diagnosis using routine diagnostic criteria. Patient demographic and clinicopathologic data were abstracted from the medical record, samples were de-identified, and their use was approved under University of Oklahoma IRB 12384. FFPE sections were mounted on Apex BOND Adhesive slides with tissue positioned within the GeoMx imaging area and baked at 60°C for at least 30 minutes. Slides were prepared on the Leica BOND RX using the GeoMx NGS RNA FFPE BOND workflow for breast tissue, including: bake and dewax preparation, ER2 heat-induced epitope retrieval for 10 minutes at 100°C, and Proteinase K digestion at 0.1 μg/mL for 15 minutes. Human Whole Transcriptome Atlas RNA probes containing UV-photocleavable barcoded oligonucleotides were hybridized overnight at 37°C. After stringent washes at 37°C (two 25-minute washes in Stringent Wash solution followed by two 2-minute washes in 2X SSC), slides were blocked in Buffer W for 30 minutes and stained for 1 hour at room temperature with the four-color morphology panel alpha smooth muscle actin (SMA)-647, SYTO 13 nuclear stain, pan-cytokeratin (CK)-532, and leukocyte (CD45)-AF594. Morphology markers guided ROI selection and segmentation of CK+, SMA+, and/or CD45+ compartments. Prepared slides were loaded onto the GeoMx DSP, each ROI was illuminated with UV light, and released oligonucleotides were collected by microcapillary aspiration into separate wells of a 96-well plate. Region-specific oligonucleotides released on the GeoMx DSP were collected into individual wells of a 96-well plate and converted into sequencing libraries according to the manufacturer's GeoMx RNA library preparation workflow for the Human Whole Transcriptome Atlas assay. Libraries were indexed according to the GeoMx protocol and sequenced as paired-end 150-bp reads on an Illumina NovaSeq X Plus instrument at the University of Colorado Anschutz Medical Campus shared-resource facility in Aurora, Colorado.