16S rRNA sequencing was performed using universal primers (27Fmod and 338R) [24 (link), 25 (link)]. Specifically, Ex Taq polymerase (Takara Bio, Shiga, Japan) was used to amplify approximately 20 ng of template DNA with Veriti Thermal Cycler (Life Technologies Japan, Tokyo, Japan) under the following cycling conditions: initial denaturation at 96 °C for 2 min, followed by 25 cycles at 96 °C for 30 s, at 55 °C for 45 s, and at 72 °C for 1 min.
The PCR product was purified with AMPure XP magnetic purification beads (Beckman Coulter, CA, USA) and quantified with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA. Samples were sequenced using a MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), according to the manufacturer’s instructions.
The PCR product was purified with AMPure XP magnetic purification beads (Beckman Coulter, CA, USA) and quantified with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). After quantification, mixed samples were prepared by pooling approximately equal amounts of each amplified DNA. Samples were sequenced using a MiSeq Reagent Kit V3 (300 × 2 cycles) and a MiSeq sequencer (Illumina, CA, USA), according to the manufacturer’s instructions.
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