16S rRNA survey of Arctic surface soil samples
DNA was extracted using the DNeasy PowerSoil kit (Qiagen) with slight modifications for the high iron content of these sediments (Emerson et al 2015, AEM). Briefly, 200 L of bead beating solution was pipetted out of each bead beating tube, then 0.25 mL of thawed sediment was pipetted with a cut-off pipette tip into the bead beating tube and 200 L of phenol-chloroform-isoamyl alcohol (PCI; 25:24:1) added after the sediment. The manufacturers protocol was followed after the addition of PCI. Extracted DNA was sent frozen to Integrated Microbiome Resources (Dalhousie University, Halifax, Nova Scotia, Canada) where it was PCR-amplified (515F/926R primer pair (V4-V5 region of the 16S rRNA gene sequence) before 250 bp paired-end Illumina sequencing) in duplicate using separate template dilutions (generally 1:1 & 1:10) using the high-fidelity Phusion polymerase. A single round of PCR is done using "fusion primers" (Illumina adaptors + indices + specific regions)with multiplexing which allows up to 380 samples to be run. PCR products are verified visually by running on a high-throughput Hamilton Nimbus Select robot using Coastal Genomics Analytical Gels. The PCR reactions from the same samples are pooled in one plate, then cleaned-up and normalized using the high-throughput Charm Biotech Just-a-Plate 96-well Normalization Kit. Up to 380 (Illumina) samples are then pooled to make one library which is then quantified fluorometrically before sequencing.