18S rDNA metabarcoding of WDL (Water in the middle of Distant Lake) sample of the Shulgan-Tash Cave.
DNA was extracted by the DNeasy PowerBiofilm Kit (Qiagen, Germany) with FastPrep-24 homogenizer (MP Biomedicals, USA) according to the protocol provided by the manufacturer. The concentration of DNA was assessed using Qubit (Invitrogen, USA) and Qubit dsDNA High Sensitivity Kit (Invitrogen, USA). The V4 region of the 18S rRNA gene was amplified with the universal eukaryotic UNonMet primers EK-565F (5'-GCAGTTAAAAAGCTCGTAGT-3') and EUK-1134R-UNonMet (5'-TTTAAGTTTCAGCCTTGCG-3') biased against Metazoa with Illumina overhang adapters. A negative sterile water control was always performed to confirm the absence of DNA contamination. The libraries were sequenced using MiSeq v3 Reagent Kit 600 cycles (Illumina, USA) on the MiSeq platform (Illumina, USA). All DNA manipulations and sequencing were performed at Joint KFU-Riken Laboratory, Kazan Federal University (Kazan, Russia).