16S rRNA V1-3 gene amplicon sequencing of pellet from human fecal sample incubation, volunteer C, T6, Glc H2O [JMF-2110-18-0022B]
Human fecal samples were freshly collected from three healthy adult individuals. Samples were transferred into an anaerobic tent (Coy Laboratory Products, USA) within 30 min after sampling. Samples were suspended in M9 medium (prepared with H2O and without glucose) to achieve a concentration of 0.1 g/ml, and further diluted 20 times in this medium. The homogenate was left to settle for 10 minutes, and the supernatant was then distributed into glass vials. An equal volume of M9 (without glucose) prepared with either D2O (99.9% atom % (at%) D; Sigma Aldrich) or H2O (for the negative control) was added to each tube, and each vial was finally supplemented with different concentrations of mucosal sugar monosaccharides (N-acetylneuraminic acid: 2 mg/ml; N-acetylglucosamine: 5 mg/ml; fucose: 2.5 mg/ml; galactose: 5 mg/ml; N-acetylgalactosamine: 2.5 mg/ml), D-glucose (5 mg/ml) or nothing (no-amendment control) (all amendment chemicals were from Sigma-Aldrich, except D(+)-galactose which was purchased from Carl Roth GmbH). After incubation for 6 h at 37C under anaerobic conditions (5% H2, 10% CO2, 85% N2), samples were pelleted and stored at -80C until further use. Amplification of bacterial and archaeal 16S rRNA genes from DNA extracts was performed with a two-step barcoding approach (UDB-H12, (https://doi.org/10.3389/fmicb.2021.669776). V1-V3 amplicons were made using the 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 534R (5'- ATTACCGCGGCTGCTGG-3') (https://doi.org/10.1111/1462-2920.13023) primers, including a 5'-head sequence for 2-step PCR barcoding, were used. {primers: AGAGTTTGATCCTGGCTCAG + ATTACCGCGGCTGCTGG, nucleic acid: DNA, flow cell identifier: JV7GK, barcoding: UDB-H12, plate-position: 2 H3, barcodes: CTCACGCAATGC + CTCACTGCTTCT}.