title: 3096-P-replete-day3-c
design: Two strains of Thalassiosira rotula (3096 and 3362) were selected to investigate the diatom  transcriptomic response to phosphate changes. Experimental cultures were established in 2-L flasks with 0.2 um filtered and autoclaved Sargasso Sea seawater adjusted to 33 PSU. All cultures were inoculated with 16-20 ml of exponentially growing cells so that the starting cell concentration was always 1000 cell per ml.  The P-replete cultures received F/2 media (Guillard and Ryther 1962, Guillard 1975) thus final concentration of phosphate was 36 uM. The P-limited cultures received 0.4 uM of phosphate and all other nutrients (nitrate, silicate, trace metals and vitamins) at the same concentration as the replete treatments. For the 3-day experiment (exponential phase harvest), all treatments were set up in triplicates. For the 9 day experiment (stationary phase harvest), there were 6-9 replicates for the P-limited treatment to ensure sufficient number of cells for RNA extractions. All cultures were incubated on a rotating platforms (135 rpm) inside incubators set to 18  C temperature and light to dark cycle of 16 and 8 hrs, respectively. Daily samples were taken to measure growth by in vivo fluorescence using 10-AU fluorometer (Turner Designs). On the day of cell collection, additional samples were taken to determine cell concentration, photosynthetic efficiency (FIRe Fluorometer System, Satlantic) and nutrient concentrations. The cells were collected by vacuum filtration on 0.22 ?m polyethersulfone filters (Express Plus Membrane, Millipore) which were flash frozen in liquid nitrogen and kept at -80  C until RNA extraction. RNA extractions were done using RNAeasy Plant DNA extraction kit (Qiagen) and quantified using Qubit HS RNA kit (Invitrogen). Eighteen RNA samples (3 per treatment) were sent for mRNA sequencing to the Duke University s Genomic Center (Table1). cDNA libraries were prepared using Kappa stranded mRNA kit (poly A enrichment method) and sequenced on one lane of the HiSeq4000 using the 2 x 150- bp chemistry.