Dataset: Thalassiosira Fe and light limitation - culture conditions
Deployment: Jenkins_2015

Thalassiosira oceanica (CCMP 1005) and T. weissflogii (CCMP 1010) isolates were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (West Boothbay Harbor, ME, USA). T. oceanica was isolated in 1958 at 33.1833°N, 65.25°W North Atlantic; T. weissflogii was isolated in 1969, at approximately 37°N 65°W Gulf Stream, between Bermuda and New York. Culture experiments were performed using a modified version of f/2 made in 0.2 µm filtered and microwave-sterilized Sargasso seawater (Guillard and Hargraves, 1993) see this dataset for modifications to f/2 for each different treatment. The inoculum for the low iron treatment came from replete cultures that had undergone two successive dilutions (1:10) into media without added iron, resulting in f/2 media with < 4 nM Fe. All macronutrient stocks were processed through a Chelex® 100 ion-exchange column (Bio-Rad Laboratories Inc., Hercules, CA, USA) containing resin prepared according to Price et al. (1989) and 0.2 µm Acrodisc® filter-sterilized (Pall Corporation, Port Washington, NY, USA). All media preparation and culture transferring was performed in a Class-100 HEPA filtered hood.

For all experiments, triplicate cultures were grown at a light level of 140 E/m2/s or 45 E/m2/s at 25 o C (click on Get Data, above) in a Percival incubator (Percival Scientific, Perry, IA, USA) and incubated gently shaking on a MaxQ® 2000 orbital shaker (Thermo Fisher Scientific, Waltham, MA, USA). Growth of the cultures was monitored daily with fluorescence measurements and cell counts (data not shown). Cultures were harvested when growth of the nutrient limited cultures began to decrease when compared to the replete cultures. Biomass was collected by gentle filtration onto 2 µm filters. Filters were placed in screw cap tubes containing 500 μL Qiagen Buffer RLT (Qiagen, Venlo, Netherlands), flash frozen in liquid nitrogen, and stored at -80°C until RNA extractions were conducted.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, with the following exceptions:  cells were lysed using 0.5 mm and 0.1 mm zirconia/silica beads (BioSpec, Bartlesville, OK, USA) mixed with the lysis buffer and bead beaten until the solution looked homogenous (approximately 1 minute). The lysis solution was then put over Qiashredder columns (Qiagen) to remove any large plant material that could clog the spin columns. To aid in the removal of DNA, two DNase digestions were performed. First, Qiagen’s RNase-free DNase Set (an on-column treatment) was used according to the manufacturer’s instructions. The second DNA removal step was conducted using the Turbo DNA-free kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA was then quantified in duplicate using the Qubit Fluorometer (Life Technologies). Following quantification, RNA samples were pooled and precipitated with a standard ammonium acetate/ethanol precipitation and sent for sequencing. An aliquot of the RNA extracted from individual replicates of the low iron and replete treatments of T. weissflogii (CCMP 1010) were also sent for sequencing.