FGF15CE412
16S rRNA Sequencing: Cecal contents were added to individual Bead plate provided by the Microbiome Core at the University of Michigan. DNA was isolated using Qiagen MagAttract PowerMicrobiome kit DNA/RNA kit (Qiagen, catalog no. 27500-4-EP) on the EpMotion 5075 (Eppendorf) liquid handler. Extracted DNA was then used to generate 16S rRNA libraries for community analysis.  The DNA libraries were prepared by the Microbiome Core as described previously 104. Briefly, DNA was PCR amplified using a set of barcoded dual-index primers specific to the V4 region of the 16S rRNA gene 105. PCR reactions are composed of 5 L of 4 M equimolar primer set, 0.15 L of AccuPrime Taq DNA High Fidelity Polymerase, 2 L of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no.12346094), 11.85 L of PCR-grade water, and 1 L of DNA template. The PCR conditions used consisted of 2 min at 95C, followed by 30 cycles of 95C for 20 seconds, 55C for 15 seconds, and 72C for 5 minutes, followed by 72C for 10 min. Each PCR reaction is normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001). The normalized reactions are pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873). The Agilent Bioanalyzer is used to confirm the size of the amplicon library (~399 bp) using a high-sensitive DNA analysis kit (catalog no. 5067-4626). Pooled amplicon library is then sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1/read 2 and index primers added to the reagent cartridge. The Preparing Libraries for Sequencing on the MiSeq (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 pM, spiked with 15% PhiX to create diversity within the run. FASTQ files are generated when the 2 x 250 bp sequencing completes.