18S rDNA metabarcoding of protistan community in saline and hypersaline continental waters of South Ural
The samples were stored in the laboratory at -80C until DNA extraction. For DNA extraction, each membrane was transferred into Lysing Matrix E tube (MP Biomedicals, LLC, Solon, OH) with addition of 2x volume of tris-saline buffer (1 Tris-l; 0,5 EDTA; 5 NaCl; MQ) and homogenized for 5 min at 50 Hz (TissueLyser LT, Qiagen). Then, membranes were enzymatically digested (lysozyme, proteinase K, SDS in total conc. 1%) and DNA was phenol-chloroform extracted (phenol-chloroform 1:1 v/v; chloroform-isoamyl alcohol 24:1 v/v) as reported previously (Gerasimova et al., 2020) [38]. Purification and desalination of DNA was carried out using DNA Clean and Concentrator-5 kit (Zymo Research, USA). Sequencing was performed using Illumina MiSeq platform and MiSeq Reagent Kit V3 2300 bp (Illumina) according to the Illumina workflow (Illumina protocol, Part #15044223, Rev. B).