18S rDNA metabarcoding of protistan community in lakes of Western Siberia
eDNA extraction, amplification and 18S rRNA gene library preparation.The samples were stored in the laboratory at 20 C until DNA extraction. DNA was extracted using the QuickDNATM Fecal/Soil Microbe MiniPrep Kit (Cat. No: D6010) according to the manufacturer's instructions, but with double DNA washing. For DNA extraction, each membrane and 100 ?l of 2x DNA/RNA Shield were transferred to a ZR Bashing Bead (0.1 & 0.5 mm) Lysis Tube (Zymo Research, California, USA, Cat. No. D6010) with addition of 750 ?l of BashingBead Buffer and homogenized for 15 min at 50 Hz (TissueLyser LT, Qiagen).The quality of the extracted DNA was checked using electrophoresis in a 1% agarose gel. The concentration of DNA was quantified with a Qubit 4 fluorometer (Invitrogen, USA) using a Qubit 1x dsDNA HS Assay Kit (Invitrogen, USA). Nucleasefree water (Qiagen, Germany Cat. No. 129115) was used as a negative control for water sample filtration.Fragments encompassing the V4 region of the 18S rRNA gene were amplified using the universal nonmetazoan primers (UNonMet):18S_EUK565F 5'GCAGTTAAAAAGCTCGTAGT3' (Simon et al., 2015);18S_EUK1134R 5'TTTAAGTTTCAGCCTTGCG3' (Bower et al., 2004).Further sequencing was performed using the Illumina MiSeq platform and MiSeq Reagent Kit V3 2?300 bp (Illumina) according to the Illumina workflow (Illumina protocol, Part #15044223, Rev. B)