18S rRNA seq: MS Estonia sample
DNA from 40-560 mg of the recovered particles was extracted using DNeasy PowerLyzer PowerSoil Kit (Qiagen, article no 12855-50). The V4 region of the eukaryotic 18S rRNA gene was amplified with 3NDF (5- ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCAAGTCTGGTGCCAG-3) and 806R (5GACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGGTATCTRATCRTCTTCG-3) primers with Illumina adapters using Phire Green Hot Start II PCR Master Mix (ThermoScientific, article no F126S), and 1 l of DNA, according to the user manual. PCR conditions were as follows: 98C for 30 s, followed by 35 cycles of 98C for 5 s, 59C for 5 s and 72C for 5 s, then 72C for 1 min, and finally 10C. PCR products were purified using AMPure XP beads (Beckman Coulter, A63880) according to the manufacturer's instructions, DNA was eluted in 30 ul of 5 mM Tris, pH 8.5. Library preparation and sequencing with Illumina MiSeq paired-end (2 x 300 bp) were performed using Eurofins NGSelect Amplicon 2nd PCR service. Eurofins processed with quality control of raw data using Cutadapt v2.17