RNA-Seq of FACS sorted – cytokine stimulated Enteric Glia cells from the LMMP after in vitro expansion

The Experiment was performed to understand the changes in expression profile of enteric glia cells in vitro to specific cytokines. Enteric glia are crucial regulators of gastrointestinal motility and immunity; however, it is not fully understood what specific cytokines can elicit a response in enteric glia. Acute T-cell activation rapidly impacts enteric glia activation, and induces cell death pathways, which negatively impacts small intestinal transit. Therefore, this experiment has been crucial to identify the specific responses of enteric glia to recombinant murine IFN?, TNFa, Il-13, Il-17A and Il-1b. In order to sequence pure enteric glia, a mouse strain of Bl6 mice with a Plp1CreERT insert for glial specificity crossed with mice with a flox controlled tdTomato fluorescent protein on the ROSA26 Locus. From these mice the longitudinal muscle layer with the myenteric plexus stripped off and enzymaticly dissociated. The enteric glia were kept as a primary cell culture and the expression of tdTomato induced with 500 nM (Z)-4-Hydroxytamoxifen (MilliporeSigma). After expansion the glia were FACS sorted and stimulated with the respective cytokine. Finally, the RNA was isolated after 24 hours and sequenced.