16S rRNA microbiome of Rattus norvegicus fecal bacteria grown in the presence of synthetic oligoribonucleotides of E. coli nature
Rat feces were sampled individually from each animal kept in a metabolic cage and stored frozen at -45 oC until use. Thawed fecal material was cultured at 37oC in GMM medium. A synthetic oligoribonucleotide fragment of leuTVPQ tRNA or its sequence derivative was added to the culture after 48 h cultivation (final concentration of 250 mkM) and after 72 h (final concentration of 500 mkM) grown until 96 h. After that bacterial cells were harvested by centrifugation, and total DNA was isolated using the EasyPure Stool Genomic DNA Kit (ThansGen Biotech, China). The sequence of 16S rRNA gene was amplified in a 50 l reaction volume from the derived DNA samples with high-fidelity Q5 DNA polymerase (New England Biolabs, USA) on a Veriti device (Thermo Fisher Scientific, USA). The obtained amplicons were purified on magnetic beads and used for preparation of sequencing libraries with Native Barcoding Kit 96 V14 (SQK-NBD114.96) (Oxford Nanopore Technologies, UK) and sequenced on PromethION (Oxford Nanopore Tech.) with FLO-PRO114M flow cells (Oxford Nanopore Tech., UK).