Durapore pvdf membrane filters

Seawater samples from the euphotic zone (between surface and 200 m) were collected at the Hawaii Ocean Time-Series Station ALOHA [Station 2 (S2)] during HOT cruise 296 (October 5–8, 2017), and at the center of a cyclonic eddy [Station 16 (S16)] during the MESO-SCOPE cruise KM1709 (June 26 to July 15, 2017) (Supplementary Figure 2 and Supplementary Table 1). The filtration was subsequently conducted through 0.22 μm Durapore PVDF membrane filters with 25 mm diameter for DNA sample collection and 47 mm diameter for lipid sample collection (EMD Millipore, Billerica, MA, United States) (see detailed information in Supplementary Table 1). Samples of 2 L were collected for DNA analysis whereas samples of 20–30 L from the remaining water were collected for lipid analysis. After filtering was completed, DNA filter samples were immediately placed in RNAlater stabilization solution (Ambion, Carlsbad, CA, United States). All filters were frozen at −80°C on board, then transported to lab using dry ice and preserved at −80°C prior to extraction of DNA and lipids.
Environmental parameters such as temperature, salinity, density and Chl a were retrieved from CTD data available on the HOT-DOGS website http://hahana.soest.hawaii.edu/hot/hot-dogs/ for station HOT296_S2 and on the SCOPE website http://scope.soest.hawaii.edu/data/mesoscope/mesoscope.html for KM1709_S16.

The mobile phase was degassed using Elmasonic sonicator (E 30 H) Elma Schmidbauer GmbH, Germany. A digital pH meter (InoLab WTW Series) was used for pH measurement and adjustment. A magnetic stirrer by Velp Scientifica, USA was used for the preparation of buffer solution and mobile phase. Filtration Assembly, Merck Millipore, Germany was utilized for filtration. The mobile phase was filtered by Durapore PVDF membrane filters with a diameter of 47mm & 0.45μm pore size, procured from Merck, Massachusetts, USA. Sample solutions were filtered through nylon syringe filters with 13mm & 25mm diameter and 0.45μm pore size, procured from CNW Technologies, Duesseldorf, Germany.

SEC-MALS was performed at 4 °C using a Superdex75 Increase 10/300GL column, a light-scattering detector (TREOS) and refractometer (Optilab rEX) from Wyatt Technology and a UV detector, HPLC pump and degasser from Jasco. System was equilibrated with 3 column volumes of SAP buffer. All buffers were filtered through 0.1 µm pore size Durapore PVDF membrane filters (Merck) following a recirculation through the system for 20 h at 0.5 mL/min to improve the baseline by removing air bubbles and particles by degasser and pre-injection filter (0.1 µm). Per measurement, 270 µg of protein in 100 µL buffer were injected and analyzed at a flow rate of 0.5 mL/min. Light scattering detector was calibrated by using monomeric BSA (Merck). The obtained signals were processed with the ASTRA software package version 5.3.4.13 (Wyatt Technology, Haverhill, UK). The concentration was determined by the refractometer using a refractive index increment dn/dc at 20.5 °C and 658 nm as calculated by SEDFIT v16.1c [86 (link)].