Synthetic community used to colonize germ-free C57BL/6 female mice - core + C. sporogenes cutC KO
Bacteria from frozen culture stocks were grown in liquid media and                 introduced as a synthetic community into female or male germ-free C57BL/6 mice fed a                 1% choline diet ad libitum. Cells were therefore 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. DNA was extracted from cecal                 samples using mechanical disruption (bead-beating with 0.1 mm zirconium beads) in                 phenol:chloroform:IAA in the same manner as described by McNulty et al. in a                 previous publication (PMID: 22030749). Libraries were prepared from extracted gDNA                 according to a slightly modified version of the protocol accompanying the Illumina                 Genomic DNA Sample Prep Kit. Briefly, total bacterial gDNA was sonicated in a                 BioRuptor XL water bath sonicator then cleaned and concentrated using a                 Macherey-Nagel PCR Purification Column before further processing. Concentrated DNA                 was blunted and poly-A tailed. A-tailed molecules were then ligated to the specified                 barcoded Illumina adapter sequences. Adapter bound DNA was size-selected on a 2%                 agarose gel by electrophoresis. Purified fragments of the desired size were PCR                 amplified and again purified over a Macherey-Nagel PCR Purification Column. Purified                 PCR products were given to the University of Wisconsin-Madison Biotechnology Center                 for quality assurance and library sequencing following the manufacturer's protocols                 for the Illumina MiSeq platform (San Diego, CA). Raw FASTQ files were provide by the                 Biotechnology Center for analysis after sequencing. Sequences were demultiplexed by                 7bp barcodes present in the adapters. Barcodes required an exact match to be                 included in community analysis (sequences without a barcode match were excluded from                 the analysis). After demultiplexing, all sequences were trimmed to 35 bases to                 eliminate low-quality bases at the ends of each read and to allow for analysis using                 Bowtie, Sequences were aligned to the reference genomes of the 6 bacteria included                 in the study using Bowtie. Only reads which mapped uniquely to the reference genomes                 were used for abundance analysis. Raw counts were normalized based on the genome                 size of each organism included in the analysis to account for genome size bias. The                 proportional representation of each organism in the analysis was determined by                 dividing its normalized counts within a sample by the total normalized counts for                 all organisms within that sample. Organisms used in the analysis include:                 Bacteroides_caccae_ATCC_43185, Bacteroides_ovatus_ATCC_8483,                 Bacteroides_thetaiotaomicron_VPI-5482, Collinsella_aerofaciens_ATCC_25986,                 Eubacterium_rectale_ATCC_33656, and                 Clostridium_sporogenes_ATCC_15579.