Transcriptomic measurements through a light:dark cycle
This dataset was downloaded from NCBI bioproject: PRJNA285666 (GEO: GSE69460).
Below detailed description was retreived from Reference: Poliner E et al., "Transcriptional coordination of physiological responses in Nannochloropsis oceanica CCMP1779 under light/dark cycles.", Plant J, 2015 Sep;83(6):1097-113.
For all experiments N. oceanica CCMP1779 cells were grown in flasks containing f/2 media under 12 h light:12 h dark cycles at 22°C under agitation (100 rpm). Light intensity during light periods was 40 μmol sec−1 m−2. ZT0–9 and ZT12–21 sets of samples were grown in two identical incubators set to reversed light/dark cycles. The ZT0 samples were collected in the dark and ZT12 samples in the light. Cell numbers were determined using a Beckman Coulter Z/2 (www.beckmancoulter.com) .
Analysis of gene expression by RNA‐Seq
For each biological replicate we used a 50 ml culture grown in a 250 ml flask (approximately 20–30 × 106 cells ml−1). Cells were collected every 3 h and two biological replicates were harvested for each time point. Cells were collected at 4000 g for 10 min at 4°C, the cell pellet was resuspended in 1.5 ml f/2 media and transferred to 2 ml tubes. The cells were centrifuged again at 18 000 g for 10 min at 4°C and flash frozen in liquid nitrogen. Samples that were in the dark at the time of collection were placed in black or amber tubes to prevent light exposure. RNA was extracted from ground frozen pellets using the Omega eZNA Plant RNA kit (Omega Biotek, www.omegabiotek.com). RNA quality was checked with the Bioanalyzer (Agilent). All samples had RINs of 6.9–7.9. Samples were sequenced at the MSU‐Research Technology Support Facility using an Illumina HiSeq 2500 (www.Illumina.com) and a single‐end 50 nucleotide run. Eight samples were sequenced in each lane using a custom bar‐coding, but the two biological replicates from the same time point were run in separate lanes. The average number of RNA‐Seq reads per sample was 23 434 157 and they ranged between 16 783 501 and 30 197 823. The reads of each of the 16 samples (eight time points, two samples each time point) were mapped to the N. oceanicaCCMP1779 V1 (Vieler et al., 2012b) genome using TopHat (Trapnell et al., 2009) with default parameters except for intron length (min 13, max 8712) and max‐multi‐hits (1). Expression level estimates in Fragments Per Kilobase of exon per million fragments mapped (FPKM) was calculated using Cufflinks (Trapnell et al., 2010) with parameter–I 8712.
Analysis of cyclic gene expression
The determination of cycling genes was performed using COSPOT (Panda et al., 2002) and the DFT as described previously (Panchy et al., 2014). In this parallel study of Chlamydomonas reinhardtii, we had compared the performance of these methods, both separately and in conjunction, using a gold standard set of cyclic genes (Panchy et al., 2014). Since no such set was available for N. oceanica CCMP1779 and the relationship between the COSPOT P‐value and ‘cyclic score’ derived from the DFT appears to be the same (Figure S13), we applied the same cutoff thresholds that we used in C. reinhardtii. A gene in N. oceanica CCMP1779 was called ‘cyclic’ if its expression vector had a COPSOT P‐value < 0.02 or a cyclic score > 0.800, which is equivalent to the P‐value of 0.02 in a population of randomized expression vectors derived from the N. oceanica FPKM data set.
Neither COSPOT nor DFT have high coverage of oscillatory expression profiles that peak only at a single time point (i.e. narrow peak of expression). To detect these patterns, we used edger (Robinson et al., 2010) to identify cases where read counts were non‐uniformly distributed across time samples. A gene was called sharply expressed if the read counts were significantly over‐expressed (P‐value < 0.05) at one time point in the light–dark cycle relative to every other time point.
Analysis of gene expression by RT‐qPCR
RNA was collected as described above and used to produce cDNA using the iScript cDNA Synthesis Kit (BioRad, www.Bio-rad.com). Primers with annealing temperatures of 55°C were designed for RT‐qPCR and checked for efficiency using serial dilutions of cDNA template and for specificity by melting curve analysis (Table S2). We used an elongation factor encoding gene 10181 as reference, since its expression was constitutive in our experiment (Data S1) and had been previously suggested to be a constitutively expressed gene in Nannochloropsis species (Cao et al., 2012). Quantitative PCR was performed using Sybr Green Master Mix (Life Technologies, www.lifetechnologies.com) and a Mastercycler®ep realplex (Eppendorf, www.eppendorf.com). Relative gene expression was obtained by the ΔCT method.