16S rRNA gene seq rat intestinal sample
DNA was extracted (PowerLyzer® PowerSoil® DNA isolation kit, MoBio 12855-100) from approx 250 mg intestinal material according to the protocol provided by the manufacturer with minor modifications. The PCR amplification of the V3-region of the 16S rRNA gene was performed by PCR reaction including fusion primers with the Ion Torrent sequencing adaptors and barcode. (PBU 5’-A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3’) and reverse primer (PBR 5’-trP1-adapter-ATTACCGCGGCTGCTGG-3’) in 20 µl total reaction volume. DNA quantity was measured using Qubit® dsDNA HS assay (InvitrogenTM, Q32851) and samples were pooled to obtain equimolar libraries containing up to 86 samples (randomized) in each library.
SUB3103199
CE.GLY5.4