Fresh-frozen tissue samples of human heart, lung, small intestine, and spleen were obtained from HuBMAP Tissue Mapping Centers (Table ). Tissue samples were collected under IRB approved protocols at each institution. Kidney samples were received as 10 µm microtome scrolls embedded in methylcellulose (each ~5 mg). All other tissue types were cut into small pieces (~5 mm) by specimen preparer at Mapping Centers. Kidney scrolls were cryopulverized in 2 mL Eppendorf Protein Lo-Bind tubes containing a 5-mm stainless steel ball (Qiagen, cat. no. 69989) with a Cryomill (Retsch, cat. no. 20.749.001) equipped with a tube adaptor. Non-kidney tissue specimen (50-100 mg) were cryopulverized with the cryomill equipped with a 25 mL grinding jar containing a 1-inch stainless steel ball. Three cycles of precooling with liquid nitrogen at 1 Hz for 3 min and grinding at 30 Hz for 1 min were performed. Pulverized tissue was transferred to a 15 mL conical tube and resuspended in 2 mL cold RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulfate (w/v), pH 7.4, 1X Halt Protease and Phosphotase Inhibitor Cocktail (Thermo Scientific)). The suspension was further disrupted by sonication on ice (40% power, cycle 2 s on, 3 s off, for 30 s total) with a probe sonicator (FisherBrand Model 120 with 1/8 inch probe) and then clarified by centrifugation (3234 × g, 30 min, 4 °C).