16S profiling of 16S_sucrose_artificial_sweetner_hcpa
Sample processing: Approximately 200 mg of fecal sample of each rat were used for DNA extraction with the commercial kit (QIAmp DNA Stool Mini kit, Qiagen, #51504) following the manufacturer's instructions. After the DNA extraction, an average of 5-10 _g of genomic material was obtained. Amplification and Next-Generation Sequencing: the hypervariable V4 region from 16S rRNA gene was amplified through PCR using genomic DNA (~ 50 ng per reaction) using the following primer pair: 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Each sample had a distinct barcode attached on the corresponding PCR product. The samples/barcodes are: ss2/CTAAGGTAAC, sn5/TACCAAGATC, ss6/CAGAAGGAAC, ss8/TTCGTGATTC, sn9/TTCCGATAAC, ss10/TGAGCGGAAC, sn15/TAGGTGGTTC, sn16/TCTAACGGAC, ss19/TCTAGAGGTC, sn20/TCTGGATGAC, ss21/TCTATTCGTC, ss25/TTAGTCGGAC, sn26/CAGATCCATC, sn27/TCGCAATTAC, ss28/TTCGAGACGC, ss30/AACCTCATTC, sn31/CCTGAGATAC, sn35/TCGACCACTC, sn36/CGAGGTTATC. (ss) indicates yogurt + sucrose fed rats. (sn) indicates yogurt + artificial sweetener fed rats. The primer sequence for the mapping file is GATGTGCCAGCNGCCGCGGTAA. The products were verified through electrophoresis in an agarose gel, purified with AMPure XP PCR Purification Kit (Beckman Coulter), quantified using Qubit dsDNA HS Assay Kit (Invitrogen) and subjected to emulsion PCR using Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Scientific). After, the resulting enriched beads were sequenced in a NGS machine (Ion Torrent PGM, Life Technologies) using Ion PGM Hi-Q View Sequencing Kit (Thermo Fisher Scientific).