Single cell RNA sequencing analysis of germ free (GF) mouse Paneth cells and microbiome-transferred exGF mouse Paneth cells.

The single cell level Paneth cell transcriptome under gnotobiotic condition has not been resolved. How gut microbiome modulates Paneth cell transcriptome at single cell level in germ-free mice was not known. We used a flow cytometry method to isolate highly pure ileal Paneth cells from germ free (GF) Paneth cell reporter mice (Lyz1-3'UTR-IRES-CreER) and from exGF Paneth cell reporter mice that were transplanted with wild type C57B/l6 mouse gut microbiota (exGF+B6M). These isolated Paneth cells were subjected to single cell RNA sequencing by 10xGenomics. Overall design: Germ-free (GF) Paneth cell reporter mice (Lyz1-3'UTR-IRES-CreER) were orally gavaged with sterile PBS to keep as germ-free controls, or orally gavaged with a freshly prepared fecal slurry collected from the cecum contents of C57B/L6 mice under anaerobic condition. 3 weeks later, mice were intraperitoneally injected with sterile tamoxifen to activate the reporter. Mice were sacrificed 2 days later when ileal Paneth cells were flow sorted for single cell RNA sequencing.