tumor stage: Normal Epithelium age and disease: Fifty-three year old man affected with primary sclerosing cholangitis
Treatment protocol
not relevant
Growth protocol
not relevant
Extracted molecule
total RNA
Extraction protocol
Fluorescently-labeled slides from the two patients were loaded onto a GeoMx machine and were scanned for selection of regions of interest (ROIs). Barcodes were collected and dispensed into a 96 wemm microtiter plate. Subsequent barcoding reading was performed on Illumina NGS platform. GeoMx NGS Pipeline was version 2.3.4. ROIs subjected to spatial transcriptomic profiling encompassed pan-cytokeratin-expressing epithelial areas of approximately 200 cells.
Label
No labeling of the barcodes
Label protocol
see extract protocol
Hybridization protocol
Spatial profiling was performed on 5 micrometer-thick formalin-fixed paraffin-embedded (FFPE) tissue sections using GeoMx technology (NanoString Technologies, Seattle, WA, USA) which was implemented by NanoString. FFPE human gallbladder slides were baked for 1 h at 65°C, and subsequently processed on Leica automation platform with a protocol that includes three steps: slide baking, antigen retrieval for 20 min at 100°C, and 1.0 microgr/ml proteinase K treatment for 15 min. After taking the slides off the Leica automation platform, each slide was incubated overnight with the GeoMx Whole Transcriptome Atlas assay probe cocktail which contains 18,677 probes (lot HWTA12002). The following day, the slides were washed and immunostained for markers of epithelium (anti-panCytokeratin), leukocytes (CD45), alpha smooth muscle actin (aSMA)-expressing mesenchymal cells, and cell nuclei (HuR), using the following antibodies: anti-CD45 (CST, #13917); anti-aSMA (AbCam, #ab202368); anti-HuR (Santa Cruz, #SC5261); anti-panCytokeratin (Novus, NBP2-33200AF488).
Scan protocol
Samples were scanned using GeoMx machine
Data processing
Raw counts were normalised using the DESeq2 method (DESeq2 Bioconductor package v1.32.0).
