Nanodrop 3300 fluorospectrometer

DNA was extracted from feces using two different methods of cell lysis and pooled as previously described by Montoya-Ciriaco et al. (2020) (link). DNA quality was verified by electrophoresis through 1% agarose gels. Amplification of the V3–V4 region of the 16S rRNA gene was performed using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-ACHVGGGTATCTAATCC-3′) primers (Herlemann et al., 2011 (link)) modified with adapters for the Illumina sequencing platform. The thermal cycling conditions of PCR were as follows: denaturation at 95°C for 2‍ ‍min, followed by 28 cycles of denaturation at 95°C for 30‍ ‍s, annealing at 55°C for 30‍ ‍s, elongation at 72°C for 30‍ ‍s, and a final extension at 72°C for 5‍ ‍min. A negative control was included in each PCR to detect reagent contamination. PCR was performed in triplicate, pooled, purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics), quantified using a NanoDrop 3300 fluorospectrometer (Thermo Fisher Scientific) with the PicoGreen dsDNA assay (Invitrogen), and combined at equal molar concentrations. Sequencing was conducted by Macrogen with 300-bp PE MiSeq runs (Illumina). Raw sequence databases are available at the Sequence Read Archive (SRA) from the NCBI under the project number PRJNA816478.

The water samples (1000 mL) were sequentially filtered through 0.45 and 0.22 µm GE® polycarbonate filter membrane. The membranes from 0.22 µm were cut into small pieces in sterilized condition and DNA extraction was performed using an optimized CTAB method (Zhou et al. 1996 (link)). The DNA extractions were performed in triplicates and replicates were later pooled prior to metagenome sequencing. DNA quality and quantity was examined with a Thermo Scientific NanoDrop 3300 Fluorospectrometer (Thermofisher Scientific, USA). The extracted DNA was randomly sheared into short fragments and ligated with Illumina adapters to construct a library. The libraries were pooled, barcoded and subsequently shotgun sequenced on one lane of a flow cell using a 150 bp paired-end run on a NovaSeq PE150 instrument (Illumina) at Novo-gene (Hong Kong). The sequences were de-multiplexed using Cassava v.2.0 and FastQC was used for quality control checks on the sequence composition of paired-end raw reads. Trimmomatic v0.36 (Q-value ≤ 38; N >10 bp; reads overlap with adapter >15 bp) was employed to remove low quality bases and any adapter contamination.

This procedure was performed as previously described (8 (link)). In brief, the total RNA from the PBMCs was extracted using a RNeasy mini kit (74104; Qiagen, Hilden, Germany) and quantified using a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using a ReverTra Ace qPCR kit (FSQ-101; Toyobo, Kagoshima, Japan) and the reverse transcription (RT) conditions were as follows: 65°C for 5 min, 37°C for 15 min and 98°C for 5 min.

Total microbial DNA was extracted from 200 mg of each fecal sample using the QIAamp Fast DNA stool mini kit (Qiagen Inc., Toronto, ON, Canada) according to manufacturer’s instructions. A bead-beating step using 300 mg of 0.1 mm zircon/silica beads was included following the addition of InhibitEX buffer and samples were agitated in a Tissuelyser II (Qiagen Inc.) for 5 min at 30 Hz. The Qiagen DNeasy Tissue kit (Qiagen Inc.) was used to extract microbial DNA from the nasopharyngeal swabs as previously detailed [17 (link)]. Briefly, this extraction method also included a 5-min bead-beating step at 30 Hz with 300 mg of 0.1 mm zircon/silica beads. The concentration of eluted DNA was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Ottawa, ON, Canada) and a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific). Negative extraction controls were also included in triplicate for both the fecal and nasopharyngeal extraction kits.

BeWo cells (3.0 × 10⁴ cells per maternal chamber) were seeded on the device and cultured for 20 h with or without medium perfusion (2 μl min⁻¹). Fetal and maternal channels were washed with balanced salt solution (BSS; 136 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 18 mM HEPES, pH 7.4), and the maternal channel including the chamber part was gently perfused with 2 mM 2-NBDG(Peptide Institute, Osaka, Japan). The device was then incubated in a CO₂ incubator for 20 min without fluid flow to enable glucose uptake in the cells. After the incubation, the BSS buffer in the fetal channel was recovered and diluted to a volume of 100 μl with BSS buffer. The fluorescence in this buffer was measured to determine glucose transfer to the fetal channel. To quantify the 2-NBDG uptakes into BeWo cells, the cells were thoroughly washed with BSS and lysed with lysis buffer (1% TritonX-100, 50 mM Tris-HCl (pH8.0), 150 mM NaCl and 1 mM EDTA). The lysate was recovered and diluted to 50 μl with the lysis buffer. Fluorescence was measured by a Nanodrop 3300 Fluorospectrometer (Thermo Scientific, Waltman, MA, USA).