rom m/z 90 to 1000 in continuum mode, and analyzed by Micromass MassLynx 4.1 software (Waters Co., USA). Peaks were also qualitatively analyzed by comparing determined molecular weights with related data in the LIPID MAPS Structure Database (LMSD).
Observation of oil bodies
To visualize oil bodies and assess their morphology, localization and numbers in the cultured cells Nile Red staining was applied (0.1 mg mL-1 in acetone in a 1:100 ratio) in dark conditions, using a similar procedure to that described for determination of the cells’ neutral lipid contents. The mixtures were thoroughly mixed, applied to a glass slide, covered with a coverslip after about 10 min, then observed under an LSM 510 META laser-scanning confocal microscope (Zeiss), with 543 nm excitation wavelength and 570–610 nm emission wavelength. Pictures were acquired randomly from at least 20 cells per sample, and typical images are presented here.
After 48 hours of N deprivation, total RNA was extracted from six replicates of control and N-deprived cultures using an RNeasy Plant Mini kit (QIAgen) following the manufacturer’s instructions. The RNA samples were treated with QIAgen RNase-free DNase I during extraction, and the extracted mRNA was enriched using oligo(dT) magnetic beads. After adding fragmentation buffer, the mRNA was fragmented into short sequences (about 200 bp), first-strand cDNA was synthesized using random hexamer-primers and the mRNA fragments as templates, and then second strands were synthesized. The resulting double-stranded cDNA was purified with a QIAquick PCR extraction kit and subjected to end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors were ligated to the fragments, the required fragments were purified by agarose gel electrophoresis, enriched by PCR amplification, and sequenced using a HiSeq™ 2000 (Illumina) instrument, with default quality parameters, at the BGI (Shenzheng), China.
Validation of RNA-Seq data by quantitative real-time PCR
Triplicate portions (2 μg) of total RNA were reverse-transcribed using random hexamer primers and an Omniscript reverse transcription kit (QIAgen). Target genes (and β-actin as a housekeeping marker) were then subjected to quantitative PCR amplification in 96-well optical reaction plates in 20 μL mixtures per well, using a SYBR Green Kit (Takara Bio) following the manufacturer’s instructions and a 7300 Sequence Detection System (Applied Biosystems/Life Technologies). The threshold cycle (Ct) for each well was measured, and the mRNA levels of the target genes in N-deprived cells, relative to those of controls, were quantified after normalization to β-actin.
RNA-Seq data processing and metabolic pathway analysis
The draft P. tricornutum reference genome was obtained from http://genome.jgi-psf.org/Phatr2/Phatr2.download.ftp.html webcite. The sequence is composed of “finished chromosomes” (Phatr2) and “unmapped sequences” (Phatr2_bd), which were annotated separately, and both were included in this study for comprehensive analysis. We aligned RNA short reads (11751810 in total) with the reference genome using SOAP 2.21 and default parameters. All original and mapped data are available at http://guanlab.ccmb.med.umich.edu/data/Yang_N_deprivation/ webcite. This resulted in 10786429 mapped reads (91.79%). Any gene with one or no reads was discarded as non-confident, and with this constraint we aligned 12312844 reads obtained from samples of N-deprived cultures, with 11326275 (91.99%) mapped to the genome. RPKM (Reads Per Kilobase per Million mapped reads) values were estimated and log2-transformed  for each gene defined in the Phatr2 database. This allowed us to estimate the fold-change in their transcript levels under nitrogen deprivation. We downloaded the KEGG pathway database (a unique set of genes that can be confidently associated with each protein in the KEGG pathway repository) released on Jun 30, 2011. We compiled all KEGG-annotated genes for P. tricornutum, and all those of other species meeting an e-value cutoff constraint of 1e-5 identified by BLAST ana