was discarded, and the collected cells were immediately flash frozen in liquid nitrogen (Figure 9 ). The use of three reactors for duplicates were necessary due to the lower cell density in the early exponential-phase. The same collection procedure was used for late exponential (Q2) and stationary phases (Q3) when cell numbers were higher except that 1?L was collected for each in duplicate. Flash frozen aliquots were stored at ?80?C until RNA extraction. Total RNA was extracted following the Spectrum Total RNA kit (Sigma Aldrich-STRN50) protocol with on-column DNAase digestion using DNase10 (Sigma-Aldrich) as suggested by the manufacturer. Initial lysis was done by a 30 second cell disruption via sonication. The protocol was performed in an RNAse free-hood to prevent RNA degradation. Total RNA was not pooled but kept as respective duplicates, and samples were sent to the National Center for Genome Resources (NCGR, Santa Fe, NM). At NCGR, mRNA was isolated twice by polyadenylation purification, and first and second strand DNA was generated with random primers. Gel products were purified and PCR enriched. Products were quantified on a Nanodrop fluorometer (Thermo Scientific) and checked for quality on a Bioanalyzer 2100 (Agilent). Using Illumina's standard protocols, flow cell construction and sequence determination was performed. Each duplicate was run on a single lane (6 total samples, 3 duplicates). Single reads were put into FASTQ format for analysis. Transcript assembly Single-end Illumina reads were analyzed using the following pipeline. First, reads from each sample were aligned against the P. tricornutum genome (v.