We will select T2D patients, among them subjects with DNA variants, and profile gene expression, expressed mutants, and proteins from sub-areas down to the level of single islets and single cells within archived pancreas FFPE tissues using a novel in situ TO-Seq assay coupled with immunohistochemistry staining of protein biomarkers, retaining the morphologic context of the profiling data. Among the set of genes measured will be biomarkers of every known molecular pathway in addition to genes associated with the development, presentation, dysregulation, and pathogenesis of T2D. By obtaining in situ gene expression profiles defining for all the cell types involved in diseased vs normal tissue within both their morphologic context and the context of genetic variants we will be able to identify molecular phenotypes and mechanisms and correlate these to cell-cell interaction, tissue homeostasis, and genetic variants. Because TO-Seq is a targeted sequencing-based assay that can measure thousands of genes, and multiplex many samples within a single sequencing run it will enable profiling data to be obtained from many separate cells at an affordable cost/cell in order to achieve the statistical power required to produce quality data supporting reliable interpretation and bioinformatics analysis. No such data exists today, and there is no system capable of producing such data on a large scale at an affordable price. Laser capture microdissection (LCM) can produce single cell profiles within their morphological context, but at a high cost which severely limits the number of samples that can be profiled. We provide preliminary data demonstrating excellent correlation and sensitivity of TO-Seq measurement of gene expression from matched fixed and frozen tissue and successful implementation of the in situ TO-Seq protocol and its integration with immunohistochemical (IHC) staining of antibody biomarkers. We demonstrate the feasibility of performing single (prostate) gland profiling with this assay. Based on these preliminary data we will optimize the in situ TO-Seq/IHC assay, implement a single islet and cell sampling system benchmarked against LCM, implement the content for the assay, and then profile the cell types present in pancreas of normal vs T2D, and between areas of pancreas exhibiting a normal vs T2D histopathology. From these data we expect to gain an understanding of the impact of genetic variant(s) at the level of gene (and protein) expression, cell interactions, and upstream regulation, and molecular mechanisms and phenotypes as they potentially contribute to disease and its progression.