Nucleopore filter

During a research cruise on the R/V Pelican on 5th May of 2010 (i.e., 15 days after the DWH blowout), oil-contaminated water samples were collected from the sea surface at 0.86 m from the site of the DWH spill (28°44.175′ N, 88°22.335′ W) in the Gulf of Mexico. At the time of collection, fractions of the samples were filtered onto 0.2-μm pore-size Nucleopore filters (Whatman, Maidstone, UK) and stored frozen for DNA extraction and barcoded 16S rRNA gene pyrosequencing, the results of which are described in detail elsewhere as the PE5 bacterial community of sea surface oil slicks [8 (link)]. A sub-sample (250 mL) of this PE5 sea surface oil slick sample was incubated at 21 °C in the dark with gentle shaking in an hermetically-sealed 250-mL wide-neck glass bottle, with a low headspace volume (ca. 100 cm³) above the water surface in order for the sample to become anaerobic over time. The sample had a visible layer of Macondo oil floating at the air–water interface—this oil formed part of the original PE5 water sample at the time of collection in the Gulf. After 4 weeks the sample incubation was manually shaken, and then 20 mL of the incubation was filtered onto a 0.2-μm pore-size Nucleopore filter and stored frozen for DNA extraction and barcoded amplicon pyrosequencing—referred to hereafter as the TG1 water sample.

At each sampling event, pre-rinsed cubitainers were used to collect approximately 5 L of surface water, of which 500 mL was filtered within 24 h of collection using a vacuum pump through two 45 mm diameter 0.2 μm pore size Nucleopore filters (Track-Etch Membrane Whatman filter). The filters were kept in sterile 15 mL centrifuge tubes at −80°C until extraction of DNA.

Surface water samples were collected along a transect from the outer (K1) to the inner part (K5) of Kongsfjorden during the Chinese Arctic Yellow River Station Expedition in July of 2011 (Fig. 1). Sampling locations, physical factors and biogeochemical properties of the samples are summarized in Table 1. After collection, 50 ml of each water sample were fixed with buffered glutaraldehyde to a final concentration of 2% for 18 h at 4 °C, then filtered onto 0.2 μm pore-sized black polycarbonate membrane (Millipore, Germany) under low vacuum (<150 mm Hg). In addition, a 50 ml water subsample of K3 fixed with buffered glutaraldehyde was filtered onto 0.2 μm pore-sized white polycarbonate membrane (Millipore, Germany). Filters were air dried and stored at −20 °C until further processing. Microorganisms present in the sample were collected by filtration of 500 ml water onto 0.2 μm pore-sized Nucleopore filters (Whatman, UK). The filters were placed in sterile 2 ml centrifuge tubes and covered with 1.5 ml lysis buffer47 (link). After processing, the tubes were immediately frozen and stored at −80 °C until DNA extraction.

Thymic lobes were isolated from E14.5 embryos and cultured for 4 days on nucleopore filters (Whatmann, NJ) placed in RPMI1640 (Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 µM 2-mercaptoethanol (Sigma-Aldrich) and 1.35 mM 2′-deoxyguanosine (2-DG, Sigma-Aldrich), as previously described^{23 (link)}. Fetal thymic lobes (2-DG FTOC) were cultured in RPMI1640-10%FBS medium and infected with lentivirus carrying miR-449a or miR-449/34 sponge expressing vectors. For in vitro differentiation, fetal thymic lobes (2-DG FTOC) were cultured with 100ng/ml recombinant human RANKL (R&D, 390-TN-010) for 4 days.

Thymic lobes were isolated from E15.5 WT and Jmjd6^−/− embryos, and were cultured for 4 days on Nucleopore filters (Whatman) placed in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Nichirei Bioscience), 50 μM 2-mercaptoethanol (Nacalai tesque), 2 mM L-glutamine (Life Technologies), 100 U ml^–1 penicillin (Life Technologies), 100 μg ml^–1 streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), MEM non-essential amino acids (Life Technologies) and 1.35 mM 2-DG (Sigma-Aldrich). After cultivation in complete RPMI medium without 2-DG for one more day, four pieces of WT and Jmjd6^−/− fetal thymi were grafted under the renal capsule of athymic (nude) and WT C57BL/6 mice. Thymic chimeras were analysed 6–10 weeks after transplantation. In some experiments, TECs were stimulated in vitro with recombinant RANKL (1 μg ml^–1; Peprotech), agonistic anti-LtβR antibody 3C8 (2 μg ml^–1; eBioscience) and/or recombinant CD40L (5 μg ml^–1; R&D systems) for 4 days before analyses.

Water samples (1 L) were collected from the six light depths (100%, 50%, 30%, 12%, 5%, and 1% of the PAR) for assessing total and size-fractionated chl-a concentrations. During the three cruises, water samples (0.3 L) were filtered using 25 mm Whatman glass fiber filters (GF/F) for the total chl-a concentration. For the size-fractionated chl-a concentration, water samples (0.7 L) were progressively filtered through Nucleopore filters with pore sizes of 20 µm and 5 µm, and Whatman GF/F filters with pore size of 0.7 µm. The filters were immediately stored in the freezer (−20 °C) until the analysis on board. After a 24 h extraction with 90% acetone, all the chl-a concentrations were quantified using a pre-calibrated Turner Designs model 10-AU fluorometer.