16S rRNA metabarcoding of DNeasy Blood & Tissue Kit
Two different DNA isolation methods were carried out depending on the origin of the samples. Prior to DNA extraction, ARS and EBS samples were passed through a 2 mm sieve to remove stones, debris or plant materials. Then, the DNA was isolated using the FastDNA SPIN Kit for Soil (MP Biomedicals) following the manufactures recommendations. Briefly, 500 mg of soil were placed into a Lysing Matrix E tube with 978 L of Sodium Phosphate Buffer, 122 L of MT Buffer and glass beads and the mechanic lysis was performed using a FastPrep-24 (MP Biomedicals) with two pulses of 40 sec. at 6 m/s, incubating on ice for 5 minutes between each pulse. To obtain the C. antarcticus DNA, a pool of 25 specimens were used for each sample using a modification of the DNeasy Blood & Tissue (Qiagen). Briefly, specimens were incubated with 180 L Buffer ATL, 20L proteinase K and 20mg of glass beads. The lysis was carried out using a FastPrep-24 (MP Biomedicals) with two pulses of 40 seg. at 6 m/s, incubating on ice for 5 minutes between each pulse. Then, the protocol was carried out according to the manufactures recommendations. DNA integrity was evaluated by 1% agarose gel electrophoresis and DNA quantity was determined by fluorometric method using the Qubit dsDNA Broad Range (BR) kit (Invitrogen). For each DNA sample, the V4 hypervariable region of the 16S rDNA gene was amplified using the primer set: 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3) (27, 28). Sequencing was performed by Novogene Co. (Beijing, China) using the Illumina NovaSeq 6000 platform in an overlapping 2250 bp configuration with a minimum throughput of 100.000 reads per sample.