IMIS

Water samples (1–2 l) were collected at selected depths through a ~30 cm diameter borehole in the ca. 4 m thick ice covers of each lake using sterile Niskin bottles. To prevent the introduction of contaminants into the lakes, precautions were taken to drill just to the surface of the water. Prior to use, each corer was rinsed properly. For each lake a different sampler was used to avoid cross contamination. In addition, we established a suitable waiting period between the drilling and the water sampling. Then, the samples were filtered through 5.0 µm Puradisc Cellulose Nitrate syringe filters (Gelman Sciences, USA) and subsequently through 0.2 µm Sterivex filters (Millipore, USA) to distinguish between particle-associated and small free-living eukaryotes. Filters were stored with 2.0 ml of Puregene lysis buffer at −80 °C until further processing and nucleic acid extraction
Study Extent: Samples were taken during the austral summers of 2011–2012 from five lake basins in the Taylor and Miers Valleys that are the focus of the McMurdo Dry Valleys Long-Term Ecological Research program (MCM LTER).
Method step description:

1. DNA from the microorganisms in the filters was extracted using a phenol-chloroform protocol. From 12 samples of the West and East lobes of Lake Bonney, we also extracted RNA using an RNeasy Mini Kit (QIAGEN, Germany). The RNA was converted to cDNA with a One-Step RT-PCR Kit (QIAGEN, Germany) according to the manufacturer’s instructions. We amplified the V7 and V8 regions of the 18S rRNA gene using primers FF390 (5′-CGATAACGAACGAGACCT-3′) and FR1 (5′-AICCATTCAATCGGTAIT-3′).
2. For the 25 µl PCR reaction, we used a proof reading enzyme (Herculase II Fusion Polymerase, Agilent Technologies, Santa Clara, USA) and 40 ng DNA (or cDNA) as a template with the following conditions; 95 °C for 3 min initial denaturation followed by 35 cycles at 95 °C for 45 s, 52 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. The 96 resulting amplicons (~350 bp) went into the library preparation for Illumina MiSeq sequencing according to the protocol presented by the Illumina customer letter for 16 S sequencing with custom primers (Illumina guide to 16 S amplicon sequencing, Part # 15044223 Rev. A) and the Nextera index kit (Illumina, San Diego, USA). The samples were sequenced on a MiSeq sequencer (Illumina, San Diego, USA) with v3 2 × 300 nt chemistry. Sequences were demultiplexed with flexbar resulting in 6.4M sequences.