16S amplicon sequencing of human gut: fecal sample
After storage at -80C, fecal samples were defrosted on ice. Aliquots of fecal material (1ml) resuspended in RNALater were centrifuged and washed once with water to remove excess fixative and salt. Then, DNA was isolated using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research) according to the manufacturer's instructions. Bead Beating was performed four times for 5 minutes each. Amplification of the V4 region (F515/R806) of the 16S rRNA gene was performed according to previously described protocols22 across two separate experiments. Briefly, 25 ng of DNA were used per PCR reaction (30 l). The PCR amplification was performed using Q5 polymerase (NEB Biolabs). The PCR conditions consisted of initial denaturation for 30s at 98C, followed by 25 cycles (10s at 98C, 20s at 55C, and 20s at 72C). Each sample was amplified in triplicates and subsequently pooled. After normalization PCR amplicons were sequenced on an Illumina MiSeq platform (PE300).