Single cell RNA sequencing of intestinal fibroblast-crypt co-cultures in organoid growth media with/without Ptger4/EP4 inhibitor (Drop-seq)

Mouse small intestinal crypts were co-cultured with primary intestinal fibroblasts in Organoid Growth Media (OGM) with/without inhibitor for the prostaglandin E2 receptor Ptger4 (EP4), labeled respectively as “Ptger4-OFF” and “Ptger4-ON” conditions. Crypts co-cultured with fibroblasts in Ptger4-ON conditions (OGM containing DMSO vehicle control) acquired a spheroid morphology. Crypts co-cultured with fibroblasts in Ptger4-OFF conditions (OGM supplemented with Ptger4 inhibitor) developed into budding organoids. Fibroblasts were isolated from the small intestine of wild type mice. For the co-cultures 2 x 10e4 fibroblasts were seeded in 48-well plates overnight. Freshly isolated crypts (n = 500) were suspended in 1:1 Matrigel (Corning, 356231) and IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) and added as an overlay on the fibroblasts. Crypts/fibroblasts were co-cultured with IntestiCult Organoid Growth Medium for 4 days. The ONO-AE3-208 Ptger4 (EP4) inhibitor (Cayman, 14522) dissolved in DMSO was added to the co-cultureson days 0 and 2 at a final concentration of 10 uM. DMSO was used as a vehicle control for the untreated co-cultures. On day 4, one pool of six Ptger4-ON co-cultures and one pool of six Ptger4-OFF co-cultures were subjected to the Drop-seq protocol. One pool of n = 6 Ptger4-ON intestinal fibroblast/crypt co-cultures (DMSO vehicle control, spheroid growth) and one pool of n = 6 Ptger4-OFF intestinal fibroblast/crypt co-cultures (ONO-AE3-208 Ptger4 inhibitor, budding organoid growth)