Illumina NovaSeq 6000 paired end sequencing; Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - primary L cells
Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - primary L cells
Primary colonic L cells from Gcg-Venus mice were collected at ZT14 by fluorescence-activated cell sorting using a BD FACS Melody cell sorter (BD Bioscience) at the University of Toronto Flow Facility. Briefly, DAPI-staining as well as side- and forward-scatter were used to remove non-Venus positive and dead cells. Venus-positive cells were isolated into LoBind tubes (Eppendorf) with RLT-Buffer (Qiagen.  Male proglucagon (Gcg)-Venus C57Bl/6J mice were used at 5-7-weeks of age. Throughout the duration of the experiment, mice were housed in a 12-hour light/12-hour dark cycle at constant room temperature with ad libitum access to food and water. Mice were fed a high-fat diet (HFD; 60% calories from fat; D12492, Research Diets) for 6 weeks to achieve a state of diet-induced obesity, with a sucrose-matched regular chow control diet (RC; 10% calories from fat; D12450J, Research Diets) used as control. A subset of animals in both diet groups was supplemented with 0.3% w/w nobiletin (NOB; R&S PharmChem. To avoid taste aversion, the nobiletin dose was gradually increased over the course of the first week Total RNA was extracted using the RNeasyPlus Micro Kit (Qiagen) with gDNA removal. NEBNext Ultra II Directional RNA-Seq + rRNA depletion (H/R/M)