Microbial mat samples were collected in the littoral or deeper parts of the euphotic zone of the lakes using a spatula or gravity corer, respectively. The upper 1 cm of the core was aseptically removed and kept dark and cool until transfer to -20°C.
Study Extent: Samples were taken from benthic microbial mats (upper 1 cm), collected in 13 lakes on the Antarctic continent, distributed across eight Antarctic regions belonging to four distinct Antarctic Conservation Biogeographical Regions (ACBR).
Quality Control: DNA concentration and quality were determined using a NanoVue spectrophotometer (GE Healthcare Life Sciences, Little Chalfont, UK). A blank DNA extraction consisting of sterile Milli-Q water was carried out in parallel.
Method step description:
- DNA was extracted from the mats using the PowerSoil DNA Isolation Kit (MOBIO Labora- tories, Carlsbad, CA, USA) according to the manufacturer’s instructions with some modifications. Tubes were agitated on a vortex for 20 extra min to ensure a good disintegration of the mats and, if not completely disintegrated, a sterile pestle was used to crush the remaining pieces.
- The cyanobacteria-specific primer set CYA359F and CYA781R(a)/CYA781R(b) was used to amplify the V3-V4 variable region of the 16S rRNA gene. PCR reactions consisted of 19 PCR buffer with 1.5 mM MgCl2, 1mg mL 1BSA, 200 uM of each dNTP, 0.2 uM of each primer, 1 U SUPER TAQ plus DNA polymerase (HT Biotechnology, Cambridge, UK), and 4 ng uL^-1 template DNA in a final volume of 50 uL. Amplification was performed using an initial denaturation step at 94°C for 2 min, followed by 30 cycles of 94°C for 45 s, 60°C (for primer 781Ra) or 57°C (for primer 781Rb) for 45 s and 68°C for 45 s, and a final elonga- tion at 68°C for 5 min. Negative controls (PCR mixes with either no DNA or the blank DNA extracts) were always included during PCR amplifications. To minimize stochastic PCR bias, amplification was carried out as six independent PCR reactions (three for each reverse primer) that were pooled before purification.
- Replicate PCR reactions were pooled and purified using the GeneJet PCR Purification Kit (Thermo Scientific, Wal- tham, MA, USA). Purified amplicons were quantified using the Quant-iT PicoGreen dsDNA Assay Kit, pooled in equimolar concentrations, and concentrated to 25 uL using the Ami- con Ultra-0.5 mL 30K device (EMD Millipore, Billerica, MA, USA). Pooled libraries were sent to Beckman Coulter Geno- mics (Takeley, UK), where primer dimers were removed using the Agencourt AMPure XP Kit (Beckman Coulter, Brea, CA, USA) and sequencing adapters were ligated to the ampli- cons. Sequences were obtained using the 454 GS FLX+ Titanium platform (454 Life Sciences, Branford, CT, USA).