title: 16S rRNA gene metabarcoding of SERF2018- 2-7 augmented sea ice middle site 2
design: Sequencing primers were 515F (Parada et al., 2016) [GTGYCAGCMGCCGCGGTAA] and 806R (Apprill et al., 2015) [GGACTACNVGGGTWTCTAAT]. We performed polymerase chain reaction in triplicate for each sample using 2x Kapa HiFi Hot Start Ready Mix in 25 _L reaction volume and 5 nmol of DNA extract.Thermocycler settings for the polymerase chain reaction were as follows: 94C for 3 min; 35 cycles of polymerase chain reaction with denaturing at 94C for 45 s, annealing at 50C for 60 s, elongation at 72C for 90s and 72C for 10 min elongation step at the end. The check of the reactions was conducted running the products on an agarose gel and checking for a product of approximately 390 base pairs. Pooled triplicate reactions were clean-up performed using Macherey-Nagel NucleoMag NGS Clean-Up and Size-Select beads.