IMIS - Marine Onderzoeksgroepen

Samples were taken aseptically, using a 30 × 30 cm colored snow squares for each sampling station.
Colored snow samples (500 mL) from Fildes Peninsula (Antarctica) taken on 2017-01-30.
Samples were allowed to naturally melt (≤ 10 °C) and were subsequently filtered through a 0.2 μm nucleopore membrane filter (Whatman). The filters were frozen at − 80 °C in cetyltrimethyl ammonium bromide (CTAB) buffer until analysis. DNA extraction was performed as described by Luo et al. (2017). PCR of microbial eukaryotes targeted the V4 region of the 18S ribosomal RNA (rRNA) gene: forward primer 3NDf (5ʹ-GGCAAGTCTGGTGCCAG-3ʹ) and reverse primer V4_euk_R2 (5ʹ-ACGGTATCTRATCRTCTTCG-3ʹ). The V4–V5 hypervariable regions of the bacte- rial 16S rRNA gene were amplified using PCR with the universal primers 515F-Y (5ʹ-GTGYCAGCMGCCGCG GTAA-3ʹ) and 926R (5ʹ-CCGYCAATTYMTTTRAG TTT-3ʹ).
PCR products were pooled and purified using a DNA gel extraction kit (Axygen, Hangzhou, China). The DNA concentration was determined using a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Germany), and quality was examined with a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, CA, USA). Finally, amplicons of all samples were pooled in equimolar concentrations and sequenced on an Illumina MiSeq2000 platform.