IMIS

Tyvek suits and nitrile gloves were worn to minimize contamination. Paleomat samples were collected from beneath ~5 cm of soil ~500 ft upshore of Lake Vanda. Sterile, DNA-free copper utensils (ashed at 550°C overnight) were used to excavate the site, and multiple sample replicates were collected, a subset of which were collected into sterile cryotubes that were immediately placed into a Taylor Wharton cryoshipper charged with liquid nitrogen, and the remainder was placed into sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA), and immediately placed on dry ice. Soil adjacent to the paleomat samples, ~20 cm away and judged by eye to be free of paleomat fragments, was collected in the same manner in multiple replicates. The modern mat sample was collected from a moat mat at the near edge of Lake Vanda (S 77° 31.168′ E 161° 38.381′), from beneath ~5 cm of meltwater. Following helicopter transport to McMurdo Station, samples were either extracted (in the case of the MiniSeq run) or maintained at a temperature of −80°C for ~1 month, then shipped on dry ice via air transport to Georgetown University, where they were maintained at a temperature of −80°C until additional processing and analysis. Because of limited access to near-shore modern mats due to ice-cover and lean biomass concentrations within the surrounding soil, sufficient amounts of high quality, high molecular weight DNA suitable for metagenomics could not be obtained, these two samples were analyzed just for amplicon-based 16S rRNA gene sequencing. Similarly, to attain μg-quantities of high molecular weight DNA from paleomat samples, several extractions from multiple replicates were pooled.
Study Extent: Samples were collected in December 2016 (austral summer), at ~11 am at Lake Vanda (S 77°31.149′ E 161° 38.315).
Quality Control: In all extraction protocols unless otherwise specified, DNA concentration, quality, molecular weight and fragment size distribution were determined via the Agilent Bioanalyzer 2100 using the HS DNA Assay (Agilent, USA). Spectrofluorometry using a Qubit High Sensitivity DNA Assay (Thermo Scientific, United States) was used to quantify the amount of gDNA present in the extractions. In both assessments the manufacturer's protocols were followed.
Method step description:

1. DNA was isolated from ancient paleomat samples collected from Lake Vanda using multiple DNA isolation protocols, using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA) or MoBio PowerSoil DNA Isolation kit (Qiagen, USA) with protocol modifications, dependent on the downstream application. Each individual extraction was performed on ~200 mg of starting material.
2. For 16S rRNA sequencing on the Illumina MiniSeq platform, DNA from mat and soil samples was extracted using physical lysis by bead-beating in the FastPrep-24 Classic Instrument (MP Biomedicals, Santa Ana, CA, USA) with a speed of 6 m/s for 40 s. Following cell lysis, DNA was purified using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA) following manufacturer's protocol. The DNA was eluted in DNase/RNase-free water and stored at −80°C. Library preparation, pooling, and MiniSeq sequencing were performed at the University of Illinois at Chicago Sequencing Core (UICSQC). Briefly, genomic DNA was PCR-amplified using a primer set targeting the V4 region of bacterial 16S rRNA gene—CS1_515F (5′- GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′)—and a two-stage PCR protocol (Bybee et al., 2011; Naqib et al., 2018). Samples were pooled in equal volume, fragments smaller than 300 bp were removed from the pooled library, which was then sequenced on the Illumina MiniSeq platform together with a phiX spike-in, and resulting in 150 bp PE reads.
3. For shotgun sequencing on the Illumina MiniSeq platform, DNA was extracted using the MoBio PowerSoil DNA Isolation kit (Qiagen, USA) according to the manufacturer's protocol, with the exception of DNA elution in 50 μL of nuclease-free water instead of the reagent provided in the kit (solution C6). The extracted DNA was stored at −80°C. A whole genome shotgun metagenomic sequencing library were constructed from 50 ng of total gDNA using the Nextera XT DNA Library Preparation Kit (Illumina USA) according to the manufacturer's instructions. The library was sequenced on the Illumina MiniSeq system using paired-end 150 bp read lengths and the MiniSeq High Output Reagent Kit (300-cycles) at the Albert P. Crary Science and Engineering Center, McMurdo Station, Antarctica.
4. An additional DNA preparation was isolated using enzymatic digestion. Briefly, paleomat samples were incubated with MetaPolyzyme (Sigma-Aldrich, USA), a mix of five enzymes (achromopeptidase, chitinase, lyticase, lysostaphin, lysozyme, and mutanolysin) designed to target bacteria and fungi (Tighe et al., 2017). For this DNA preparation, the bead-beating step was omitted to maximize the yield of high molecular weight DNA. DNA extraction from cell lysates was carried out using a ZymoBIOMICS DNA Miniprep Kit following manufacturer's protocol, and the resulting DNA was eluted in DNAase/RNase-free water and stored at −80°C. DNA from this set of extractions was used for whole genome shotgun using the whole DNA fraction, as well as size-selected (>2,500-bp) gDNA fraction. The sequencing libraries were prepared using the MiSeq Reagent Kit v3 according to the manufacturer's instructions, and were sequenced at UICSQC on Illumina MiSeq, producing 2 × 300 bp reads.
5. DNA was isolated for Pacbio sequencing using enzymatic digestion, and the ZymoBIOMICS DNA Miniprep Kit following manufacturer's protocol with modifications as described above. The sequencing library was prepared following the Pacific Biosciences 2 kb SMRTBell Template Preparation and Sequencing protocol with an additional bead-based template cleanup using 0.5X AMPure PB beads to eliminate smaller-sized fragments. A final library QC was performed with a Qubit fluorometer to determine library concentration and an Agilent Bioanalyzer 2100 (Agilent Genomics) using the DNA 12000 reagent kit to determine the size distribution of the library. The prepared sequence library was loaded onto a single Sequel v2.1 SMRTcell at a concentration of 6 pM, following the PacBio diffusion loading protocol and including a polymerase-bound complex cleanup. One 600-min movie was taken of the SMRTcell.)
6. Total RNA was isolated from ~500 mg of ancient paleomat material collected from Lake Vanda (WA3A) using the Zymo Direct-zol RNA MiniPrep kit (Zymo Research Corp., USA) according to the manufacturer's instructions. Briefly, the paleomat material was mechanically disrupted on dry ice in the original collection tube using a sealed, disposable RNase-free spatula. TRIzol reagent (500 μl) was added directly to the sample, which was subsequently vortexed and centrifuged at 12,000 × g for 30 s to remove particulates, and the supernatant was transferred to an RNAse-free tube. Cell lysis in TRIzol was repeated for the remaining paleomat material to provide a total of 1,000 μl supernatant, which was then mixed with one volume of 100% ethanol and loaded onto a Zymo-Spin IIC Column. The RNA sample was treated in-column with 5 U of DNase I, washed and eluted in 20 ul of RNase-free water. RNA yield and integrity were determined via fluorometric quantitation using the Qubit RNA HS Assay Kit (Thermo Scientific, United States), and the Agilent Bioanalyzer 2100 using the RNA 6000 Nano Kit (Agilent, USA). Sequencing libraries were constructed from 2 ng of total RNA using the SMARTer Stranded Total RNA-Seq Kit v2-Pico library preparation kit (Takara Bio, USA) according to the manufacturer's instructions using protocol option 2 (without prior RNA fragmentation). Library preparation included both an RT (-) control (using paleomat RNA as template, but excluding the SMARTScribe Reverse Transcriptase enzyme), and a negative control using water as template. Both controls resulted in no library construction, confirming the absence of any contaminating gDNA. The final RNASeq (cDNA) library was sequenced on the Illumina MiSeq System using paired-end 300 bp read lengths and the MiSeq Reagent Kit v3 (600-cycle) (Illumina, Inc, USA) at the Genomics and Epigenomics Shared Resource at Georgetown University Medical Center.