NaNO3, 0.17 mM CaCl2, 0.30 mM MgSO4, 0.43 mM K2HPO4, 1.00 mM KH2PO4, 0.43 mM NaCl, 0.17 mM EDTA, 18 mM FeSO4, 0.18 mM H3BO3, 61 mM ZnSO4, 15 mM MnCl2, 10 mM MoO3, 13 mM CuSO4, and 3.3 mM CoNO3. Cultures were maintained at 25uC61uC, with continuous (24 hr) white fluores- cent light illumination (200 mE m22 s21). Cultures were supple- mented with 2% CO2/air and mixed with a magnetic stir bar at 500 rpm. To induce nitrogen deprivation, rapidly growing cells were centrifuged for 5 min at 5,0006 g, washed once in nitrogen- free mBBM, and resuspended in nitrogen-free mBBM for continued growth. Cell growth was monitored via cell count measurements using a 0.1 mm depth, Hy Lite_hemocytometer (Hausser Scientific). Cultures were inoculated at a cell density of approximately 3.56106 cells/mL. Fatty Acid Methyl Ester (FAME) analysis Total fatty acid content was determined via transesterification of glycerolipids followed by GC-FID analysis, using a method adapted from Lepage and Roy [33]. Briefly, 50 mL samples of cell culture at 7.856107 cells/mL (nitrogen replete) and 5.006108 cells/mL (nitrogen deplete) were harvested via centrifugation for 5 min at 5,0006 g. Cell pellets were quenched in liquid nitrogen and lyophilized overnight. Approximately 5 mg of dry biomass was suspended in chloroform-methanol (2:1, v/v), and glyceroli- pids were transesterified in HCl-methanol (5%, v/v) for 1 h at 85uC in the presence of a tridecanoic acid methyl ester as an internal standard (Sigma Aldrich). Fatty acid methyl esters were extracted in hexane (Sigma Aldrich) at room temperature for 1 hr and analyzed by GC-FID. For all FAME analyses, three replicates were examined. Fluorescence Microscopy Non-polar lipid accumulation was qualitatively examined using epifluorescence microscopy. Cells were harvested from nitrogen replete and nitrogen deplete media, as described above. Cells were treated with 10% DMSO (to increase membrane permeability) and stained for five minutes with 10 mg/ml of the non-polar lipid fluorescent dye, BODIPY 493/503 (Molecular Probes, Invitrogen Corporation). Cells were immobilized on microscope coverslips by mixing with 1% low-melting-temperature agarose (heated to 65uC to solubilize) in a 1:1 ratio. Images were acquired using a Nikon Eclipse 80i Epifluorescent microscope. Chlorophyll autofluores- cence was detected using a 660/50 band-pass optical filter, and BODIPY 493/503 fluorescence was detected using a 525/50 band-pass filter. Isolation of mRNA A 50-mL sample of a nitrogen replete cell culture was harvested via centrifugation for 2 minutes at 3,000 6g. Cell pellets were resuspended in 5 mL RNeasy Lysis Buffer (Buffer RLT, Qiagen, RNeasy Plant Mini Kit), frozen in liquid nitrogen, and ground into a fine powder by mortar and pestle on liquid nitrogen. RNA was extracted using a Qiagen RNeasy Plant Mini Kit with glass bead disruption. The lysate was divided among 10 microfuge tubes and eluted into RNase-free ddH20. A Qiagen RNeasy MinElute Cleanup Kit was utilized to concentrate the RNA. Isolation of soluble protein fraction Cells were harvested at cell densities of 7.856107 cells/mL (nitrogen replete) and 5.006108 cells/mL (nitrogen deplete) via centrifugation for 2 minutes at 3,0006 g. Cell pellets were immediately quenched in liquid nitrogen, thawed and solubilized on ice in 2 mL of lysis buffer (50 mM Tris, pH 8.0, 150 mM Systems Biology Analysis of Chlorella vulgaris NaCl, 1 mM DTT, 10% glycerol, supplemented with 16 cOmplete Protease Inhibitor Cocktail solution (Roche Diagnostics Corporation, Indianapolis, IN)). The cells were then sonicated on ice at 4uC, at 90% power setting for 30 seconds66 cycles, with a one minute cool-down period between sonication cycles using a Braun-Sonic-L ultrasonicator. Lysates were cleared via two cycles of centrifugation at 16,0006 g at 4uC for 30 minutes, and the supernatants were isolated for use in subsequent proteomic analysis. Transcriptome Analysis Total RNA was first assessed for quality on a Bioanalyzer 2100 using Nano 6000 LabChip (Agilent Inc., Santa Clara, CA). A complementary DNA (cDNA) sequencing library was prepared from the total RNA using a mRNA-seq Sample Preparation Kit (Illumina). Briefly, poly A+ RNA was isolated from 10 mg total RNA using Sera-Mag Magnetic Oligo-dT beads. Purified mRNA was fragmented, annealed to high concentrations of random hexamers, and reverse transcribed. Following second strand cDNA synthesis, end repair, and A-tailing, oligo adapters complementary to sequencing primers were ligated to cDNA fragment ends. Resultant cDNA libraries were size fractionated on an agarose gel by excising 200-bp fragments followed by amplification with 15 cycles of polymerase chain reaction. Clusters were generated from the cDNA sequencing library on the surface of a flowcell in the Cluster Station (Illumina) by bridge amplification. The flowcell was used to perform 56 cycles of sequencing-by-synthesis chemistry in the Genome Analyzer II. The manufacturer’s Genome Analysis OLB 1.8 pipeline (Illumina, San Diego, CA, USA) was used to perform image analysis (Firecrest), and base calling (Bustard). Short nucleotide reads obtained via Illumina sequencing were assembled by the Velvet software [34] to produce error-free, unique contiguous sequences (contigs). The Oases program [35] was then utilized to cluster the contigs in the preliminary Velvet assembly into small groups (loci), and construct transcript isoforms for each of these loci. For the assembly of contigs using Velvet, we chose a k-mer length of 25 that maximized the average length of the transcript isoforms that constituted the output from the Oases program. Chlorophyta nucleotide sequences were downloaded from the NCBI Gene database and formatted using the makeblastdb program from the standalone BLAST+ program suite in order to obtain a nucleotide database compatible for BLAST analysis. Transcript isoforms were annotated by the local alignment of assembled transcript sequences against this Chlorophyta nucleotide database using the standalone NCBI BLAST+ program suite. Nucleotide query sequences of the transcript isoforms were locally aligned against the nucleotide sequences in the database using the nucleotide blast (blastn) program from the standalone BLAST+ program suite, and the results from this nucleotide BLAST+ search enabled the assignment of gene models to these transcripts. The nucleotide blast search was complemented by the local alignment of the six-frame conceptual translation products of the query transcript sequences against a formatted database of viridiplantae protein sequences downloaded from the RefSeq protein database using the blastx program. Gene ontology enrichment was performed on the annotated transcriptome and the subset of the transcriptome matching the C. vulgaris proteome utilizing the Blast2GO software version 2.4.8 [36]. Proteome Analysis Gel-based liquid chromatography-mass spectrometry (GeLC/ MS) was employed for comparative shotgun proteomic analysis. 20 mg soluble proteins were separated using one-dimensional SDS- PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25851