Wistar male rat feces
The amplification of the extracted DNA was done by PCR using the primers, 16S Amplicon PCR forward primer:5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, and 16S Amplicon PCR Reverse Primer: 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHV GGGTATCTAATCC targeting the V3 and V4 hypervariable regions of the bacterial 16S rRNA gene. Every single PCR was performed in 25 L reaction volumes of which 12.5 L was 2X KAPA HiFi Hotstart ready mix (KAPA Biosystems, Woburn, MA, USA). The rest corresponded to 5 L of each forward and reverse primers (1 M) and 2.5 L of extracted DNA (10 ng). The cycling conditions were also the same for each PCR: initial denaturation at 95 C for 3 min, followed by cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and elongation at 72 C for 30 s, with a final extension at 72 C for 5 min. AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) were used for the PCR clean-up to purify the 16S V3 and V4 amplicon away from free primers and primer-dimer species. After this, the index PCR was performed under 95 C for 3 min; 8 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for 30 138 s; 72 C for 5 min, and hold at 4 C, using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) to attach dual indices and Illumina sequencing adapters. Then, before the quantification, the pooled PCR products were purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA). The resultant amplicons were sequenced at MiSeq (Illumina, USA), using a paired-end (2x300nt) Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA) (Pastor-Villaescusa et al., 2021; Plaza-Diaz et al., 2021; Plaza-Diaz et al., 2019).