GeoMx Digital Spatial Profiler Immuno-oncology (IO) Proteome Atlas (IPA) Assay for Human Pancreas v1

Purpose: This protocol details processing fixed paraffin sections from human pancreas for the Nanostring GeoMX digital spatial profiler (DSP) immuno-oncology proteome atlas (IPA). This technology is based upon a defined antibody panel of 575 markers. Each antibody is conjugated to an RNA probe. The RNA probes are designed with UV cleavable linkers and DSP barcodes. Fluorescent-labeled antibodies against antigens that define specific cell types were used as morphology markers. Morphology markers are used to identify cell types of interest with nuclear counterstaining permitting tissue spatial information. Sample regions of interest (ROI) are defined based on the three morphology markers and each ROI is illuminated with UV light thereby cleaving hybridized RNA probes. Cleaved probes are aspirated via microcapillary and deposited into a unique well of a 96-well collection plate. The collection plate is used for next generation sequencing (NGS). Scope: This document was written following GeoMX NGS guidelines (university.nanostring.com) with minor modifications for the University of Florida Molecular Pathology Core. The entire workflow is described for manual slide staining and GeoMX DSP instrument use. Steps for library construction, sequencing, bioinformatics, and data analysis are in outline form only. Library preparation and sequencing are outsourced to the University of Florida ICBR facility. Expected outcome: Targeted-protein quantification via bulk RNA-sequencing will be obtained from fixed human pancreas sections within regions of interest and subareas defined by cell morphology markers.