title: 16s rDNA amplicon of mouse feces (carnitine group)
design: All fecal samples were aliquoted and stored at -80 degrees Celcius for further examination. Fecal genomic DNA was extracted using the QIAamp PowerFecal DNA Kit according to the manufacturer's instructions. Two-step polymerase chain reaction (PCR) workflow was performed for library preparation under procedures described in the Illumina 16S sample preparation guide. The 16S rRNA gene V3-V4 region was amplified and sequenced on the Illumina Miseq platform for paired-end sequencing (2 x 300 bps). The 16S rRNA gene V3-V4 region was amplified using a primer overhanging adapter (forward = 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 and reverse = 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3). Dual indices and Illumina sequencing adapters were attached through PCR by using a Nextera XT Index Kit according to the manufacturers instructions. After each PCR process, PCR cleanup was performed using AMPure XP beads to purify V3-V4 amplicon from the free primer and primer dimer. The sizes of PCR products were verified using the Bioanalyzer DNA 1000 chip. Library quantification was performed for quality control before sequencing by using the Agilent Technologies 2100 Bioanalyzer. The pooled libraries were then sequenced on the Illumina Miseq platform with v3 reagents for paired-end sequencing (2  300 bps).