Global transcriptomic changes following N-deprivation.
This dataset was downloaded from NCBI under bioproject: PRJNA182180 (GEO: GSE42508).
Below detailed description was retreived from Reference: Li J et al., "Choreography of Transcriptomes and Lipidomes of Nannochloropsis Reveals the Mechanisms of Oil Synthesis in Microalgae.", Plant Cell, 2014 Apr;26(4):1645-1665.
Design of Experimental Strategy
Nannochloropsis oceanica IMET1 was inoculated into modified f/2 liquid medium, which was prepared with 35 g L−1 sea salt, 1 g L−1 NaNO3, 67 mg L−1 NaH2PO4*H2O, 3.65 mg L−1FeCl3*6H2O, 4.37 mg L−1 Na2EDTA*2H2O, trace metal mix (0.0196 mg L−1 CuSO4*5H2O, 0.0126 mg L−1 NaMoO4*2H2O, 0.044 mg L−1 ZnSO4*7H2O, 0.01 mg L−1 CoCl2, and 0.36 mg L−1MnCl2*4H2O), and vitamin mix (2.5 µg L−1 VB12, 2.5 µg L−1 biotin, and 0.5 µg L−1 thiamine HCl). The algal cells were grown in liquid cultures under continuous light (∼50 µmol photons m−2 s−1) at 25°C and aerated by bubbling with a mixture of 1.5% CO2 in air. Approximately 5 liters of algal culture in column photobioreactors was harvested by centrifugation at the linear growth phase (OD750 = 2.6). Cell pellets were washed three times with axenic seawater and inoculated into N-free f/2 medium (800 mL, n = 3) and f/2 medium (800 mL, n = 3) in triplicates, respectively. Cultures started with the same initial cell concentration of OD750 = 2.6 and were exposed to continuous illumination of 50 μmol m−2 s−1. Cell aliquots were collected for RNA isolation after being transferred to the designated conditions for 3, 4, 6, 12, 24, and 48 h. Three biological replicates of algal cultures were established under each of the above N+ and N- conditions, respectively.
Transcriptome Sampling and Sequencing
Total algal RNA under the above conditions was extracted using Trizol reagents (Invitrogen). For mRNA-Seq, the poly(A)-containing mRNA molecules were purified using Sera-mag Magnetic Oligo(dT) Beads (Thermo Scientific) and were fragmented into 200- to 300-bp fragments by incubation in RNA fragmentation reagent (Ambion) according to the manufacturer’s instructions. The fragmented mRNA was then purified away from the fragmentation buffer using Agencourt RNAClean beads (Beckman Coulter). The purified, fragmented mRNA was converted into double-stranded cDNA using the SuperScript double-stranded cDNA synthesis kit (Invitrogen) by priming with random hexamers. Strand nonspecific transcriptome libraries were prepared using the NEBNext mRNA Library Prep Reagent Set (New England Biolabs) and sequenced for 2×90-bp runs (paired-end) using Illumina HiSequation 2000.
To ensure quality, the raw data (2×90-bp paired-end reads) were modified as follows: First, adapter pollutions in reads were deleted, and second, because the sequence qualities of Illumina reads degrade quickly toward the 3′ end, all reads were trimmed from the 3′ end until the 3′-end–most position with Phred-equivalent score was 20 or greater. The raw data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus with the reference series number GSE42508. These filtered Illumina reads were aligned to the N. oceanica draft genome with TopHat (version 2.0.4, allowed no more than two segment mismatches) (Trapnell et al., 2009). Reads mapped to more than one location were excluded. Third, the short read mapping results from TopHat were used for the differential gene expression analysis with Cufflinks (version 2.0.4), as described below (Trapnell et al., 2010, 2013).
Estimation of Differential Gene Expression
For each of the mRNA-Seq data sets under each experimental condition, gene expression was measured as the numbers of aligned reads to annotated genes by Cufflinks (version 2.0.4) and normalized to FPKM values.
Genes were considered to be significantly differentially expressed if either of the conditions was met: (1) Their expression values showed at least a 2-fold change with a false discovery rate (FDR)-corrected P value ≤ 0.05 (Benjamini-Hochberg correction, which is provided by the Cuffdiff program in the Cufflinks package, version 2.0.4) between control and stressed conditions for at least one time point, and moreover their FPKM values at either conditions were ≥ 10. (2) Their expression values showed a 1.5- to <2-fold change with an FDR-adjusted P value ≤ 0.05 between control and stressed conditions for at least two time points, and moreover their FPKM values at either of the conditions were ≥10.
The 3255 differentially expressed genes were grouped into 16 clusters based on their temporal expression patterns by the k-means clustering using the MultiExperiment Viewer 4.8 (MeV4.8; http://www.tm4.org/mev.html) with Euclidean distance (Saeed et al., 2006). The optimal number of clusters was identified and investigated by performing a figure of merit analysis within MeV4.8 (Yeung et al., 2001). Figure of merit analysis showed that the value was stabilized after a partitioning into 12 to 18 clusters using k-means algorithm. Therefore, the transcripts were split into 16 clusters, each of which exhibits a particular pattern of temporal dynamics.
Validation Using Real-Time PCR
To further test the validity of the mRNA-Seq results, RNA extracted from the same cultures for mRNA-Seq was subjected to the PrimeScript RT reagent kit with gDNA Eraser (Takara) for cDNA synthesis. Also, qRT-PCR was performed by standard methods (Roche) as previously described (Guénin et al., 2009). Ct values were determined for triplicate independent technical experiments performed on triplicate biological cultures (n = 3). Relative fold differences were calculated based on the ΔCt method using the actin amplification product as an internal standard. Primer pairs used for qRT-PCR analyses are listed in Supplemental Table 2. Sizes of amplification products were 100 to 300 bp. The coefficient of determination between the qPCR results and the mRNA-Seq results was 0.94 (R2) (Supplemental Figure 3).
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