fluorescence intensity (?) with respect to cell number (?) (A). Nile Red fluorescence intensity indicating the increase in lipids is shown with the depletion of external nitrate (?) and phosphate ( ? ) (B) . Phosphate concentrations are multiplied by a factor of 10 for scaling purposes (e.g., 0.2 mM = 0.02 mM). Arrows indicate time points at which cells were harvested for RNA sequencing. Analysis of RNA-sequence data A time course assessment strategy was employed to identify transcripts that were differentially expressed during growth and lipid accumulation from early-exponential (Q1), late-exponential (Q2), and stationary-phase cells (Q3) in conjunction with pH, nutrient availability, light, DIC, cell number, protein, carbohydrate, and lipid. Cultures were sampled in duplicate for each time point, total RNA extracted, and each sample sequenced via an individual lane of Illumina sequence determination. RNA-sequence analysis was used to globally monitor gene expression during nutrient-depletion and lipid accumulation under the tested growth conditions. Using TopHat and Cufflinks (see Materials and Methods), the transcript relative abundance was calculated and reported as FPKMs (Fragments Per Kilobase of exon per Million fragments mapped), a normalized quantity that is directly proportional to transcript abundance as recently reported [ 27 - 29 ]. To compare transcript levels between the time points, Q2 and Q3 were reported as the relative ratio compared to Q1. The Q1 time point was considered the basal transcript level condition in which cells were growing exponentially under nutrient-replete conditions with low lipid levels. Based upon work in bacteria, transcript and