Mixer hc

Colonoids used for sequencing are described in Table S1. UBD was diluted in organoid expansion media, then colonoids in Matrigel were treated overnight (18 h) with $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ UBD, with control colonoids receiving the same volume/volume of organoid expansion medium as the vehicle. Dosage was chosen based on prior studies^{26 (link),48 (link)–51 (link)} that showed molecular changes to cells or animals exposed to uranium, but with minimal cytotoxic damage, since acute uranium exposure is not linked to cytotoxicity. Next day, colonoids were harvested from Matrigel using Cultrex Organoid Harvesting Solution, then resuspended in $500\mathrm{\mu }\mathrm{L}$ TrypLE enzyme solution (ThermoFisher), transferred to a 24-well plate and spun at $600\text{\hspace{0.17em}rpm}$ at 37°C for 45 min in a spinoculator (Mixer HC; USA Scientific) to digest into single cells. Single cells were washed in $10\mathrm{mL}$ of Advanced DMEM/F12, pelleted again, resuspended in $1\mathrm{mL}$ of organoid expansion media and filtered with $40\text{-}\mathrm{\mu }\mathrm{m}$ Flowmi cell strainers (SP Bel-Art). Cells were transported to the UNM Analytical and Translational Genomics Core in the UNM Comprehensive Cancer Center for counting and processing. Average processing time from colonoids to library preparation was $<2.5$ hours, and the cells were highly viable ( $>90\%$ ).