title: Female mouse 4 16S: Choline+IMC
design: Cells were maintained over the duration of the experiment within mice until ex vivo sampling occurred. Immediately after collection, cecal samples were subjected to flash-freezing and stored at -80C until total community DNA extractions could be performed. Microbial DNA was extracted following MO BIO PowerSoil-htp 96 Well Soil DNA Isolation Kit procedure with an additional incubation at 65C for 10 min prior to plate shaking. The 16S rRNA V4 hypervariable region was amplified with barcoded primers (Caporaso et al. 2012) in triplicate using the 5 PRIME HotMasterMix (VWR). Products were quantified with Quant-iTTM PicoGreen dsDNA Assay Kit (Thermo Fisher) and samples were combined in equal amounts (~250 ng per sample) to be purified with the UltraClean PCR Clean-Up Kit (MO BIO). Pooled amplicons were sequenced on the Illumina HiSeq platform to generate single-end reads. Raw sequences were processed with the open source Quantitative Insights Into Microbial Ecology (QIIME) software package version 1.9.1 (Caporaso et al. 2010) to produce de-multiplexed and quality controlled sequences. Samples were binned into OTUs using the pick_closed_reference_otus.py script at 97% similarity using SUMACLUST (Kopylova et al. 2016) against a Greengenes reference database (McDonald et al. 2012).