6XPPDU\$EVWUDFW 3URMHFW The goal of the KIDney Organ Specific Project (KIDOSP) is to generate comprehensive multimodal and multiscalar single cell and spatial data of the adult human kidney spanning micro, meso and macro scales with a focus on resident functional tissue units (FTU). These maps spanning 11 different data outputs from 9 technologies will be used for integrated analysis by the KIDney Data Analysis Core (KIDDAC) to generate a high resolution and comprehensive integrated atlas for the KIDney Single cell and Spatial Molecular Atlas Project (KIDSSMAP). Implementation of KIDOSP requires robust, highly quality-controlled protocols applied to different regions of the kidney that economizes tissue usage and enables interrogation by multiple orthogonal methods at different scales. The KidOSP team is uniquely poised to meet these challenges and generate a multimodal and multiscalar atlas through an established infrastructure. This includes enrolment of patients from multiple sources that permit broad sharing of genomic data, controlled preanalytical parameters and careful processing and preservation of samples in a manner compatible with all the assays to be performed. To ensure that the information is captured at the cell, FTU, region, organ and whole-body level, registration in spatial coordinates is necessary during the life cycle of the specimen. Diversity in regions sampled across lifespan, sex and race are needed for the atlas to be broadly usable. The technologies generating data include paired single nucleus chromatin accessibility and RNA expression (snRNA/ATAC-seq -cell type and state diversity), smFISH and DART-FISH (targeted high resolution spatial interrogation of 30-1000 transcripts), spatial transcriptomics (untargeted genome wide spatial mapping of dissociative technologies), CODEX (multiplexed spatial protein interrogation to bridge with RNA technologies and 3D IF technologies), 3D multiplexed immunofluorescence (3D IF-subcellular resolution to define neighborhoods in micro-FTUs), lightsheet fluorescence microscopy (LSFM-anatomical maps at mesoscale of key and 3D maps of neurovascular associations with FTUs) and Scattering Raman Spectroscopy (SRS-2D, 3D label free volumetric mapping at subcellular scale). Our team's work in multiple national consortia and in the setup phase of HuBMAP have established the key tissue processing methods and quality control pipelines needed to generate multimodal, multiscalar data at a single-cell resolution and in spatial contexts. These will be processed through an analytical pipeline in the data analysis core (DAC) to build a comprehensive high-resolution spatial atlas of the kidney. The protocols established are applicable to human clinical disease biopsies, extensible to new technologies, and adaptable by other sites, and will become a great resource, through HuBMAP, for the community.