IMIS

The ice cores were drilled with a trace-metal-clean (electropolished steel) ice auger, 14 cm in diameter. Two ice cores were collected and pooled at each station for the microbiological analyses. We emphasised careful sampling and subsequent sample processing to avoid contamination. The ice cores collected were cut with an ethanol-wiped handsaw into two to seven pieces, depending on the ice thickness (each horizon 10–30 cm), crushed and placed in sterile plastic containers at 4 °C over night in darkness after which the rest of the ice was quickly melted in a water bath with constant stirring. The melted samples were immediately filtered after becoming fully melted.

Study Extent: The samples were collected from 10 pack-ice stations along the Weddell and Lazarev Seas during the Antarctic Winter Ecosystem Climate Study (AWECS, leg ANT-XXIX/6)-expedition aboard the R/V Polarstern during the austral winter in June–July 2013.

Method step description:

1. For the DNA extractions, approximately 500 ml of the melted sea-ice were filtered onto sterile 0.22-μm membrane filters (Ø 47 mm; Whatman GE Healthcare, Little Chalfont, Kent, UK) and frozen in liquid nitrogen and later transferred to –80 °C.
2. The bacterial community DNA was extracted from the filters with a PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc, Carlsbad, CA, USA), as described by Eronen-Rasimus et al. (2014), 6 months after the cruise. In addition to the samples, negative controls without the sample were extracted.
3. The 16S ribosomal RNA gene region from V1 to part of the V3 was amplified with a polymerase chain reaction, using the universal bacterial primers F8 and R492. A two-step polymerase chain reaction and Illumina MiSeq (Illumina Inc, San Diego, CA, USA) paired-end multiplex sequencing were performed at the Institute of Biotechnology, University of Helsinki, Finland.