16SrRNA in vitro gut model
Samples from the BL, and the EPs were used for metataxonomy by 16S rRNA MiSeq sequencing (Illumina Inc, San Diego, CA, USA). Considering metataxonomy, bacterial diversity was obtained by the library preparation and sequencing of the 16S rRNA gene. The following two amplification steps were performed: an initial PCR amplification using 16S locus-specific PCR primers (16S-341F 5-CCTACGGGNGGCWGCAG-3 and 16S-805R 5-GACTACHVGGGTATCTAATCC-3) and a subsequent amplification integrating relevant flow-cell-binding domains (5-TCGTCG GCAGCGTCAGATGTGTATAAGAGACAG-3 for the forward primer and 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3 for the reverse overhang), and lastly unique indices selected among those available Nextera XT Index Kits were combined according to manufacturers instructions (Illumina Inc, USA). Both input and final libraries were quantified by Qubit 2.0 Fluorometer (Invitrogen, USA). In addition, libraries were quality-tested by Agilent 2100 Bioanalyzer High Sensitivity DNA assay (Agilent technologies, Santa Clara, CA, USA). Libraries were sequenced in a MiSeq (Illumina Inc, USA) in the paired end with 300-bp read length (Marino et al. 2019). Sequencing was conducted by IGA Technology Service Srl (Udine, Italy)