2.1. Samples
The study included ten sediment samples from different deep-sea areas
(Appendix 1, Table 1), totaling five stations: four in the Atlantic
Ocean and one in the Mediterranean Sea (see Fig. 1, Table 1).
Standardized sampling was performed using a Multicorer or Pushcorer
(both with cores of 20 cm2) and slicing under sterile
conditions. For this study, only the first two layers of sediment (0-1
cm and 1-3 cm) were used for each station. Samples were frozen at -80°C
immediately after recovery and slicing for later extraction. DNA
extraction was performed using ~10 g of substrate with
the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) following the
manufacturer’s protocols. Extraction controls were included (empty
sampling bags conditioned onboard and rinsed with water and manufacturer
tubes initially filled with sterile water instead of sediment). Final
extracts were concentrated three times with 70% ethanol for
precipitation reaction and diluted in molecular grade water.