title: 16S rDNA-seq of cardiovascular disease patients with vegetarian-diet intervention gut metagenome: fecal sample, month 38
design: Fecal samples were transported to our lab in cold storage (4-7 C) within 24 hours of collection. The feces were then aliquoted and stored at -80 C for microbiome analysis and other experiments. Fecal genomic DNA was extracted by using the Mobio PowerFecal DNA Isolation Kit according to the manufacturers instructions and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). A two-step polymerase chain reaction (PCR) workflow was conducted for library preparation in accordance with procedures described in the Illumina 16S sample preparation guide. The 16S rRNA gene V3-V4 region was amplified using a primer overhanging adapter (forward = 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGC CTACGGGNGGCWGCAG-3 and reverse = 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAG ACAGGACTACHVGGGTATCTAATCC-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).