GSM6797406: gp91.42.1; Homo sapiens; OTHER
DNA was extracted from stool samples using PowerSoil® DNA Isolation Kit (MO BIO Laboratories, #12888-100) as per manufacturer's instructions (available online at https://mobio.com/media/wysiwyg/pdfs/protocols/12888.pdf), with the following 2 modifications: 1) Prior to bead beating, proteinase K (20μL) (Qiagen, #19131) was added to each sample, and samples were incubated for 10 minutes at 65C° using a heating block, and 2) the bead beating step was completed using a TissueLyser II (Qiagen) at 30 Hz for 2 minutes. DNA was eluted in UltraClean® PCR Water (MoBio Laboratories, #17000-10). The stool microbiome was characterized by amplicon sequencing of the 16S rRNA gene's V4 hypervariable region amplified using the following primers (515F = GTGCCAGCAGCCGCGGTAA and 806R = GGACTACCAGGGTATCTAAT) that were modified to include a linker sequence, a 12 bp index sequence and a heterogeneity spacer as described in [56]. The PCR conditions were: sample DNA (2.5μL), 10X PCR Buffer (2.5μL), dNTP Mix (4μL), Takara LA Taq Polymerase (0.25μL) (Takara Bio USA, #RR013A), PCR Water (13.75μL) (Qiagen, #17000-10), and 1μL of each primer (515F (10μM) and 806R (10μM)). Reactions were performed in duplicate according to the following conditions: 98C° for 30 seconds, 30 cycles (98C° for 15 seconds, 58C° for 15 seconds, 72C° for 15 seconds), and 72C° for 60 seconds. Libraries were combined, cleaned up with the Agencourt AMPure XP Kit (Beckman Coulter, #A63880) and quantified using the Quant-IT dsDNA High-Sensitivity Assay Kit (Invitrogen, #Q33120) as above. Normalized libraries were then pooled, purified using a Qiagen MinElute column (Qiagen, #28004), and sequenced using a 250-bp paired-end sequencing protocol on the Illumina MiSeq platform (Illumina Inc.) at the NIH Intramural Sequencing Center (NISC, NIH, Rockville, MD). OTHER: 16S rRNA sequencing