16S rRNA V4 gene amplicon sequencing of Mouse 414, small intestine contents, control diet [JMF-1904-2-0051A]
Amplicon sequencing of Mouse 414, small intestine contents, control diet. Flow cytometry was used to quantify the absolute number of bacterial cells per sample for small intestine, cecum and fecal pellets using FACS Melody and the FACS_Chorus software (BD, Germany). Samples were dissolved in 500 ul PBS and then strained through a 40 um cell strainer (Corning, USA). After filtering, 5 ul SYBR green was added which was later used for gating samples. Absolute counting beads (CountBright, Invitrogen, ThermoFisher Scientific, Austria) were used for cell counts according to the manufacturer's instructions. Each sample was counted in triplicate and the mean total cell counts g-1 were calculated. DNA from intestinal contents was extracted using the FastStool DNA extraction kit automated on a QIACube Connect, following the manufacturer's instructions. Thereafter, amplicons were generated using primers targeting the V4 region (515F Parada [5'-GTG YCA GCM GCC GCG GTA A-3'] and 806R Apprill [5'-GGA CTA CNV GGG TWT CTA AT-3'] of bacterial and archaeal 16S rRNA genes, barcoded in a unique dual setup, and sequenced on the Illumina MiSeq system as further described by Pjevac et al. (https://doi.org/10.3389/fmicb.2021.669776.). {primers: GTGYCAGCMGCCGCGGTAA + GGACTACNVGGGTWTCTAAT, nucleic acid: DNA, flow cell identifier: CBM45, barcoding: CDB-H12, plate-position: 17 G9, barcodes: CACAAGTATCGA + AACTTTCAGGAG}.