 was shaken and centrifuged, and the upper phase was discarded. Then 2 ml water was added to wash the extract, the mixture was centrifuged (12000 rpm for 10 min) and the upper phase was removed. Finally, the organic solvents were evaporated by nitrogen and the residues were stored in a −80°C refrigerator until analysis. Total lipid extracts were separated into lipid classes according to Demandre et al. with modifications . The dried algal lipid extracts were re-dissolved in 1 mL buffer A (isopropanol-hexane, 4:3), and the solutions were centrifuged at 14000 rpm for 10 min to remove solid impurities, then transferred to a glass vial. Lipid classes were subsequently separated by HPLC using a silica column and a mobile phase consisting of a 20-min linear gradient from 100% solution A to 100% B (isopropanol-hexane-water, 8:6:1.5), followed by isocratic solution B for a further 25 min, with the column and sample collection system set at 20°C. The total running time, including a 5 min re-equilibration step, was about 50 min. The flow rate was 1.0 mL min-1 and 10 μL samples were injected. Eluting fractions of lipids were detected spectrophotometrically at 205 nm, manually collected in separate glass tubes, then stored at −20°C. Compounds in the lipid classes fractionated were diluted in isopropanol and identified using a Micromass Q-TOF micro system (Waters Co., UK) equipped with a syringe pump, LockSpray and ESI interface, operated in positive ionization mode. The mass spectrometer conditions were as follows: electrospray capillary voltage, 3.2 kV; ion source temperature, 110°C; desolvation temperature, 350°C; cone voltage, 15V; cone and collision gases, nitrogen and argon, at 60 L h-1 and 600 L h-1 flow rates, respectively. Sodium formate solution was used to calibrate the TOF mass spectrometer in positive electrospray ionization (ESI+) mode, and leucine enkephalin (L-EK) was used as an external mass calibration standard. Masses were scanned from 100 to 1100 amu at approximately 0.4 scan s-1, and data were collected from 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 