title: Illumina NovaSeq 6000 sequencing; Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - mGLUTag L cells
design: Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - mGLUTag L cells
library_construction_protocol: Total RNA was extracted at t=8 hr from synchronized murine GLUTag cells (reference here: 10.1007/978-3-319-16104-4_21) after treatment with vehicle or palmitate, with and without nobiletin. Cells were grown on 6-well plates in Dulbecco's modified Eagle's medium with 25 mmol/L glucose and 10% FBS for 48-hours.  Cells were pre-treated for 12-hours with vehicle or 0.5 mM palmitate (PAL; Sigma-Aldrich) made by dissolving in water at 70°C and then adding to growth medium. Cells were also treated with vehicle or 20 μM nobiletin (R&S PharmChem and Sigma-Aldrich; dissolved in DMSO). Cells were synchronized by incubation for 12-hours in 0.5% FBS media followed by addition of 20 μM forskolin in 10% FBS media for 1-hour. Media was then changed to regular growing media with the appropriate treatments for an additional 8-hours.  Total RNA was extracted using the RNeasy Plus Mini Kit with QIAshredder (Qiagen). Takara SMART-Seq v4 PLUS Kit (SSv4 PLUS)