title: V4-SSU RNA gene, K2 station, 6 m depth
design: One liter seawater sample was collected at 6 m depth at the K2 stations (CTDEEP), filtered through 2 ľm polycarbonate filter (Millpore). DNA was extracted using Power Water DNA isolation kit (MoBio). Three PCR reactions were carried out in 25 ľl volume using 30 ng of DNA, 0.4 uM costume design Miseq primers, Phusion polymerase master mix (Thermo Scientific). The cycling conditions were: initial denature step at 98şC for 30 s, 30 amplification cycles of 98şC for 10 s, 50şC for 20 s and 72şC for 40 s, followed by a final step of 10 min at 72şC. Bands were cut out and purified using a gel extraction kit (Thermo Scientific). The three PCR reactions were pooled and quantified using the Qubit broad range assay (Life Technologies). Samples were diluted to 4 nM concentration and pooled into two multiplexed libraries, then sequenced on using the MiseqNano kit (v.2), at 2 times 250 cycles, following the manufacturer's protocol, with 10% of PhiX DNA added as an internal control (MiseqNano, Illumina).
library_name: miseq17