Protocol for isolating cells from a single cryopreserved gastrointestinal endoscopic biopsy for single-cell RNA sequencing.

Techniques exist to isolate cells from fresh intestinal tissue; however, approaches for recovering viable cells from cryopreserved duodenal and colonic specimens are lacking. Here, we present a protocol for isolating cells from a single cryopreserved gastrointestinal endoscopic biopsy for single-cell RNA sequencing (scRNA-seq). We describe steps for thawing and washing cryopreserved endoscopic biopsies, enzymatic digestion of biopsies, and purification preparation. We then detail procedures for dead cell removal, quantification of cell recovery, and 10X Gel Bead-in-Emulsion (GEM) generation and library preparation. For complete details on the use and execution of this protocol, please refer to Richards et al.1.