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Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging ofdoi:10.1016/j.xpro.2021.100597 pmid:34169291 pmcid:PMC8209738 fatcat:4kjpewgz6nb6bmditrqfnnx7xy