biostudies-arrayexpressMiguel Gonzalez AceraMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14879The 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 IFNγ, TNFα, 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.biostudies-arrayexpressSequencing - The clustering of the index-coded samples was performed according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generatedSample Collection - 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.Library Construction - 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.Nucleic Acid Extraction - Total RNA was extracted from the samples taken from colons of mice using the peqGOLD total RNA kit (Peqlab/VWR) following the manufacturers' instructions.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Normalization of the data was performed by DESeq2 using the mean of ratios.Sequence Alignment - Sequence mapping and alignment was performed using STAR v2.7.10bUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAMus musculusMarvin BubeckJay PatankarMiguel Gonzalez AcerafalseRNA-Seq of FACS sorted – cytokine stimulated Enteric Glia cells from the LMMP after in vitro expansionThe 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 IFNγ, TNFα, 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.2025-07-01T00:00:00Z2025-03-03T09:25:25.501Z2025-03-03T09:25:25.501ZE-MTAB-14879ERP169746EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184