ITS2 metabarcoding of protistan community in saline and hypersaline continental waters
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). Purification and desalination of DNA was carried out using DNA Clean and Concentrator-5 kit (Zymo Research, USA). The ITS2 region of the rRNA locus was amplified using ITS3-2024F (5-GAT GAA GAA CGY AGY RAA-3) and ITS4-2409R (5-TCC TCC GCT TAT TGA TAT GC-3) primers (White et al., 1990). The libraries containing  ITS2 region were sequenced on the MiSeq platform using MiSeq Reagent Kit v3 (600-cycles) (Illumina).