ena-STUDY-INSTITUT DE CIENCIES DEL MAR-05-06-2018-16:05:57:145-134
16S rDNA genes from different size-fraction across vertical profiles in the global ocean sampled during the Malaspina 2010 expedition
We selected a total of eight stations of those sampled during the Malaspina 2010 Expedition  between December 2010 and July 2011. The selected stations were distributed across the global tropical and subtropical ocean (latitudes between 30° N and 40° S): three in the Atlantic Ocean, two in the Indian Ocean, and three in the Pacific Ocean. At each station, four depths were sampled corresponding to the surface (SFC, 3 m), the deep chlorophyll maximum (DCM, 48–150 m), the mesope- lagic (MESO, 250–670 m), and the bathypelagic waters (BATHY, 3,105– 4,000 m). Surface water was sampled with a Niskin bottle, and water from the other depths was sampled with a rosette of Niskin bottles attached to a conductivity–temperature–depth (CTD) profiler. Vertical profiles of salinity, potential temperature, and dissolved oxygen were recorded continuously with the CTD sensors installed in the rosette sampler.  Prokaryotic biomass from different size fractions was collected by prefiltering the water through a 200-µm net mesh and sequentially filtering 10 L through 20-, 5.0-, 3.0-, 0.8-, and 0.2-µm pore-size filters, all 47-mm polycarbonate filters (20-µm pore-size filter from GE Water and Process Technologies and the rest of the filters from Millipore), using a peristaltic pump, resulting in five different size fractions (0.2–0.8, 0.8–3.0, 3.0–5.0, 5.0– 20, and 20–200 µm). Filter clogging or particle dislodging may affect the taxonomic composition observed in each size fraction, but we tried to minimize these issues by filtering at very low speed and pressure and by changing frequently the filters. The filters were flash-frozen in liquid N2 and stored at -80 °C until DNA extraction. The DNA was extracted with a phenol-chloroform protocol. The hypervariable V4–V5 region of the 16S rRNA gene was PCR amplified with primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) and sequenced in an Illumina MiSeq platform using 2 × 250 bp paired-end approach at the Research and Testing Laboratory facility (rtlgenomics.com/)   See Further details in: Mestre M, Ruiz-Gonzalez C, Logares R, Duarte CM, Gasol JM, Sala MM. Sinking particles promote vertical connectivity in the ocean microbiome. Proc Natl Acad Sci U S A. 2018;115: E6799-E807. https://www.pnas.org/content/pnas/115/29/E6799.full.pdf
We selected a total of eight stations of those sampled during the Malaspina 2010 Expedition  between December 2010 and July 2011. The selected stations were distributed across the global tropical and subtropical ocean (latitudes between 30° N and 40° S): three in the Atlantic Ocean, two in the Indian Ocean, and three in the Pacific Ocean. At each station, four depths were sampled corresponding to the surface (SFC, 3 m), the deep chlorophyll maximum (DCM, 48–150 m), the mesope- lagic (MESO, 250–670 m), and the bathypelagic waters (BATHY, 3,105– 4,000 m). Surface water was sampled with a Niskin bottle, and water from the other depths was sampled with a rosette of Niskin bottles attached to a conductivity–temperature–depth (CTD) profiler. Vertical profiles of salinity, potential temperature, and dissolved oxygen were recorded continuously with the CTD sensors installed in the rosette sampler.  Prokaryotic biomass from different size fractions was collected by prefiltering the water through a 200-µm net mesh and sequentially filtering 10 L through 20-, 5.0-, 3.0-, 0.8-, and 0.2-µm pore-size filters, all 47-mm polycarbonate filters (20-µm pore-size filter from GE Water and Process Technologies and the rest of the filters from Millipore), using a peristaltic pump, resulting in five different size fractions (0.2–0.8, 0.8–3.0, 3.0–5.0, 5.0– 20, and 20–200 µm). Filter clogging or particle dislodging may affect the taxonomic composition observed in each size fraction, but we tried to minimize these issues by filtering at very low speed and pressure and by changing frequently the filters. The filters were flash-frozen in liquid N2 and stored at -80 °C until DNA extraction. The DNA was extracted with a phenol-chloroform protocol. The hypervariable V4–V5 region of the 16S rRNA gene was PCR amplified with primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) and sequenced in an Illumina MiSeq platform using 2 × 250 bp paired-end approach at the Research and Testing Laboratory facility (rtlgenomics.com/)   See Further details in: Mestre M, Ruiz-Gonzalez C, Logares R, Duarte CM, Gasol JM, Sala MM. Sinking particles promote vertical connectivity in the ocean microbiome. Proc Natl Acad Sci U S A. 2018;115: E6799-E807. https://www.pnas.org/content/pnas/115/29/E6799.full.pdf