title: Illumina MiSeq paired end sequencing; Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - 16S
design: Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion - 16S
library_construction_protocol: Fecal pellets were collected from the colon. 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 (CON; 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 (R&S PharmChem). DNA was extracted from samples using the ZymoBIOMICS DNA miniprep kit (ZymoBIOMICS).  The V4 hypervariable region of the 16S rRNA gene was amplified using uniquely barcoded 515F (forward) and 806R (reverse) sequencing primers to allow for multiplexing (Caporaso et al., 2012). Amplification reactions were performed using 12.5 uL of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems), 1.5 uL of 10 uM forward and reverse primers, 7.5 uL of sterile water and 2 uL of DNA. The V4 region was amplified by cycling the reaction at 95°C for 3 minutes, 18x cycles of 95°C for 15 seconds, 50°C for 15 seconds and 72°C for 15 seconds, followed by a 5-minute 72°C extension. All amplification reactions were done in duplicate to reduce amplification bias, pooled, and checked on a 1% agarose TBE gel. Pooled duplicates were quantified using PicoGreen and combined by even concentrations.