Crypt isolation and single‐cell dissociation

The duodenum or ileum of tamoxifen‐injected Lyz1^{3′UTR‐CreER}; R26R‐tdT mice was harvested, opened longitudinally, and cut into smaller fragments. The pieces were rinsed in cold PBS before shaking in 1× HBSS Ca/Mg‐free (Sigma, Catalog No. H9394), containing 10 mM EDTA (Invitrogen, AM9260G) for 10 min at 4°C. The tissues were then transferred to fresh 10 mM EDTA‐HBSS medium for 30 min at 4°C. After vigorous shaking, the flow‐through collected from 70‐μm filtering was centrifuged at 700 g for 5 min at 4°C. Next, the pellet was resuspended with TrypLE Express (Thermofisher, 12605028) containing 0.5 mg/ml of DNaseI (Qiagen, 79254) for no more than 18 min at 37°C. Next, the cell suspension was passed through a 40‐μm filter, centrifuged at 700 g for 5 min at 4°C, and resuspended in FACS buffer (F12/DMEM containing 2 μM ROCK inhibitor, 2% BSA, 2% FBS, and 2 mM EDTA).