title: 18S rDNA metabarcoding of protistan community in the Volga river
design: For DNA extraction, each membrane filter with 100 l of DNA/RNA Shield was transferred into Lysing Matrix E tube (MP Biomedicals, LLC, Solon, OH, USA) with addition of 2x volume of tris-saline buffer (1 Tris-l; 0,5M EDTA; 5 NaCl; MQ) and homogenized for 5 min at 50 Hz (TissueLyser LT, Qiagen). Then, membranes were consequently enzymatically digested with lysozyme (10 mg/ml, 1/10 v/v) at 37C for 30 min and with proteinase K and 10% SDS (in total conc. 1%) at 60C for 30 min. DNA was extracted using the phenol-chloroform method (phenol-chloroform 1:1 v/v; chloroform-isoamyl alcohol 24:1 v/v) as reported previously (Gerasimova, 2020; Gerasimova, 2023). Purification and desalination of DNA was carried out using DNA Clean and Concentrator-5 kit (Zymo Research, USA). The final quality of the DNA was checked using electrophoresis in 1% agarose gel (in 1x TAE buffer, BioRad). Concentration of DNA was quantified with a Qubit 4 fluorometer (Invitrogen, USA) using a Qubit 1x dsDNA HS Assay Kit (Invitrogen, USA). The nuclease free water (Qiagen, Germany) was taken as negative control of water sample filtration, and ZymoBIOMICS (ZymoResearch, USA) was used as positive control of high-throughput sequencing.The 18S rRNA gene V4 region of ca. 550 bp was PCR amplified with UNonMet primers set EK-565F 5'-GCAGTTAAAAAGCTCGTAGT (Simon et al.,2014) and EUK-1134R-UNonMet 5'-TTTAAGTTTCAGCCTTGCG biased against Metazoa (Bower et al., 2004) with Illumina overhang adapters. The 18S amplicons were processing according to the Illumina workflow (Illumina protocol, Part #15044223, Rev. B) and sequenced with MiSeq platform (Illumina) using V3 Reagent Kit generating 2300 nucleotide reads.