RNA-Seq of FACS sorted – cytokine stimulated Enteric Glia cells from the LMMP after in vitro expansion
RNA-Seq of FACS sorted – cytokine stimulated Enteric Glia cells from the LMMP after in vitro expansion
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.  Total RNA was extracted from the samples taken from colons of mice using the peqGOLD total RNA kit (Peqlab/VWR) following the manufacturers' instructions. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.