Analysis of intestinal crypt, villus, and polyp cells from WT and Vil1-Grem1 with or without anti-Grem1 antibody treatment by scRNA-seq
Analysis of intestinal crypt, villus, and polyp cells from WT and Vil1-Grem1 with or without anti-Grem1 antibody treatment by scRNA-seq
The proximal small bowel with dissected out and opened longitudinally.  The muscle layer was scraped off mechanically with a coverslip and discarded.  The villus compartment and crypt compartment were separated mechanically and digested using the MACS human umbilical cord dissociation kit (Miltenyi, 130-105-737) following the manufacturerâ€™s instructions, with an enzymatic digestion time of 1h15m.  The 3 mice per group were pooled.  Cells were stained with propidium iodide (1:500, Invitrogen) and live cells were sorted using the BD FACSAriaTM Fusion Cell Sorter. Vil1-Grem1 mice were treated with a novel anti-Grem1 antibody (UCB Ab7326 mIgG, UCB Pharma), 30 mg/kg subcutaneously weekly from the age of 6-weeks old for 10 weeks. Nucleic acids were directly processed on intact cells using the Chromium Single Cell 3' Reagent v3 chemistry kit (10X Genomics) on a Chromium Instrument (10x Genomics). Libraries were prepared using the Chromium Single Cell 3' Reagent v3 chemistry kit (10X Genomics) on a Chromium Instrument (10X Genomics). Library quality was assessed using a TapeStation (Agilent Technologies).