PROJECT SUMMARY ABSTRACT: Overall The small bowel and colon are organs critical for maintaining homeostasis of the human body by mediating nutritional absorption upon the ingestion of food. Though both organs are extensive in length, there are known differences in function and cellular heterogeneity within different portions of each. Also, a cross section anywhere in the bowel reveals a complex layering of components involved in absorption and secretion, motility of gut contents, circulation, and immunity. In this submission, we propose to continue our efforts in the Stanford Tissue Mapping Center (TMC) to produce multi-modal, three-dimensional single-cell resolution maps of the small bowel and colonic wall structure. This will serve as a community resource to further study intestinal function and disease. We will collect tissues from deceased organ donors with explicit consent for distribution among the HuBMAP consortium and broad access genome data sharing (GDS). Three sets of technologies will then be employed. Tissue samples will be subjected to single-nuclei ATAC-seq and RNA-seq. These open chromatin and transcriptomic profiles will be spatially mapped to tissue sections using the CODEX (CO-Detection by indEXing) multiplexed spatial immunoassay. We will also employ the Molecular Cartography multiplexed fluorescence in situ hybridization (FISH) assay to enable more accurate integration of CODEX and single-nuclei data. The resultant three- dimensional maps will span all layers of the bowel wall and include the epithelial, enteroendocrine, vascular, lymphatic, nervous, immune, and muscular cell populations that contribute to normal bowel function.