IMIS

1 L of surface sea water from each station was collected and prefiltered through a 20-µm mesh sieve to remove most of the mesozooplankton and large particles, and then directly filtered through a 0.2-µm pore size nucleopore membrane filter (Whatman). The filters were frozen at −80 °C in cetyltrimethylammonium bromide (CTAB) buffer until laboratory experiments were carried out.
Study Extent: Samples were taken in January 2013, during the 29th Chinese National Antarctic Research Expedition at Greatwall Cove and Ardley Cove (king George Island, Antarctica).
Method step description:

1. DNA extraction was performed as described by Luo et al. (2009).
2. Polymerase chain reaction (PCR) was performed using primers with barcodes flanking the hypervariable V4 region of the 18S rRNA gene: 3NDf with the reverse primer V4_euk_R2. PCR was conducted in 20 μL reactions with 0.2 μM each of the primers, ~10 ng of template DNA, 1 × PCR buffer, and 2.5 U of Pfu DNA Polymerase (Promega, USA). The amplification programme consisted of an initial denaturation step at 95 °C for 2 min, followed by 30 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension of 72 °C for 5 min. PCR products were pooled and purified using a DNA gel extraction kit (Axygen, Hangzhou, China). The DNA concentration of each PCR product was determined using a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Germany) and was quality controlled on a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, CA, USA). Finally, amplicons of all samples were pooled in equimolar concentrations.
3. 18S rRNA amplification and sequencing on the Illumina MiSeq2000 were done by following the standard protocols of Earth Microbiome Project (EMP) (Caporaso et al. 2012).