Seawater Metagenome Sampling and DNA Extraction

Samples for metagenome analyses were collected from St. 301 (5 m deep) and St. 307 (5, 30, 150, and 750 m deep) in October 2019 (coordinates 43.155517 N 28.005383 E and 43.1696 N and 29.001283 E, respectively). Up to 6.9 L of seawater from each sampling depth were filtered through a series of 20 μm Nylon Net filters (Millipore), 5 μm polycarbonate membrane filters (Millipore), and 0.22 μm SterivexTM Filter Units (Merck). DNA was then extracted only from these Sterivex filters using standard phenol-chloroform protocol [43 (link)]. In short, Sterivex filters were treated with CTAB lysis buffer and then treated with 1 mg ml^− 1 lysozyme and 0.2 mg ml^− 1 proteinase K (final concentrations). Then nucleic acids were purified with phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol.