Invitrogen qubit 4.0 fluorometer

Total RNA was prepared from InnovaPrep Wet Foam Elution using QIAamp Viral RNA minikit, as mentioned previously. To serve as an in situ control for DNA sequencing, the ZymoBIOMICS High Microbial Load Spike-in Control I (Zymo Research, Irvine, CA, USA) was added to each sample, per manufacturer’s specifications. Total DNA was prepared from 250 μL of InnovaPrep Wet Foam Elution using the DNeasy PowerSoil Pro Kit (Qiagen, Germantown, MD, USA), following the manufacturer’s instructions for use on the automated QIAcube Connect platform. Nucleic acid concentrations were determined using Qubit Broad Range Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA) on an Invitrogen Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) for RNA and double-stranded DNA, respectively.

The V1-V2 hypervariable regions of 16S rRNA gene sequences were amplified by PCR using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA) and primers from the Nextera XT Index Kit (Illumina, San Diego, CA, USA)^{50 (link)}. Amplification was performed on a Veriti Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). PCR products (approximately 450 bp) were visualized by agarose gel electrophoresis, purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA), and DNA concentrations were measured using a NanoDrop ND1000 spectrophotometer and Invitrogen Qubit 4.0 Fluorometer (Thermo Fisher Scientific). Tag-indexed samples were diluted and pooled into a low DNA-binding tube in equal amounts from each sample. DNA concentration in the pooled samples was confirmed by qPCR, using KAPA SYBR FAST qPCR MasterMix (KAPA Biosystems). The library was applied to a MiSeq sequencing platform (Illumina). Nucleotide sequence data reported are available in the DNA Data Bank of Japan (DDBJ) Sequenced Read Archive under the accession number DRA010713.