2.3. Microbiome Profiling of In Vitro and Human Study Samples

The primers S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′)/S-D-Bact-0785-a-A-21 (5′GACTACHVGGGTATC TAATC C-3′) 5′ [42] were used to amplify the V3/V4 variable region of the 16S rRNA gene for the profiling of the bacterial fecal microbiota using the Phusion High-Fidelity PCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA). After PCR product purification, the Illumina MiSeq system protocol was used for library preparation. Indexing PCR was performed to amply the dual-index barcodes to the amplicon (Nextera XT V.2 Index Kits; Illumina, San Diego, CA, USA). The purification of the barcoded amplicons was performed with the Agencourt AMPure XP-PCR Purification system (Beckman Coutler, Inc., Brea, CA, USA). The Qubit dsDNA HS Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) was used to quantify the products. Equal concentrations of all the purified amplicons were pooled into a library that was sequenced (2 × 300 bp) on a MiSeq Illumina platform in the Teagasc Food Research Centre sequencing facility (Teagasc Moorepark, Fermoy, Ireland). ENA accession number: PRJEB39031.