16S V4 of Homo sapiens: gut
Approximately 200 mg of stool sample was mixed with 1.2 mL of stool lysis buffer and homogenized by bead beating using lysing matrix E (MP Biomedicals, SP, Brazil) and Disruptor Genie (Scientific Industries, NY, USA). The homogenized suspension was incubated at 95C for 10 minutes and centrifuged at maximum speed for 1 minute and 20 seconds. The supernatant was collected and the remaining protocol was followed according to the manufacturer's instructions. The DNA extracted was eluted in a buffer solution from the PSP kit and stored at -20C. DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA). The V4 region of the 16S rRNA gene was PCR-amplified using the AccuPrime Taq DNA Polymerase (Invitrogen, CA, USA). The amplicons were purified with AmPure beads (Beckman Coulter, CA, USA), quantified by Quant-iT PicoGreen dsDNA kit (Thermo Scientific, MA, USA), libraries were pooled, and sequenced on the MiSeq platform (Illumina, Inc., USA), using MiSeq Reagent Kit V2.