Determination of the whole mRNA transcriptome of the three morphotypes of Phaeodactylum tricornutum by RNA-sequencing

We performed RNA-sequencing experiments to analyse the differential gene expression in the three morphotypes of Phaeodactylum tricornutum Pt3 strain. P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, oval and triradiate. Currently, little information is available regarding the molecular and cellular biology of P. tricornutum and especially the physiological significance of its morphogenesis as well as its mechanism of regulation. In this study, we adapted the P. tricornutum Pt3 strain to obtain algal cultures particularly enriched in one dominant morphotype: fusiform, triradiate or oval in order to determine their whole mRNA transcriptome. The diatom cells were grown at 19°C in 1L bioreactors on a 16h/8h light night cycle. The intensity of the light used was 68 µmol.m-2.s-1. The nutritive medium was composed of natural seawater 100% for the fusiform and triradiate morphotypes and 10% for the oval morphotype, enriched in Conway containing 80 mg.L-1 of sodium metasilicate (Na2SiO3). The diatom cells were cultured under ambient air at a regulated flow rate of 0.83 L.min-1 CO2. Bioreactors were inoculated with a final microalgae concentration of 1x106 cells.mL-1. According to this protocol, the cells follow similar growth curves and the culture conditions used in this study allow us to maintain the specificity of each culture batch as well as the diatom cell integrity and organization for each morphotype. The cultures were done in parallel. Four biological replicates for each culture were prepared for RNA extraction. The cells were harvested in the middle of the exponential growth phase at day 8 to proceed with the RNA purification. Total RNA was isolated from 8.108 cells. The cells were recovered by centrifugation and immediately resuspended in 1mL of TRIzol® Reagent (Ambion by Life Technologies).The suspension was then transferred in a 2 mL tube of lysing matrix E (MP Biomedicals) and immediately flash frozen in liquid nitrogen. The samples were then stored at -80°C until RNA extraction. For RNA purification, cells were lysed by using the FastPrep®-24 high-speed benchtop homogenizer (MP Biomedicals). Then, RNA was isolated by using the standard TRIzol® reagent protocol given by the supplier (Ambion®, LifeTechnlogies) followed by a purification on a Nucleospin RNA II column (Macherey-Nagel) including an on-column DNAse I treatment. For each preparation, the RNA concentration was determined in duplicate using a Nanodrop spectrophotometer (Thermo Scientific). In addition, the RNA quality was controlled using an Agilent 2100 Bioanalyser (Agilent Technologies).Libraries were then prepared from 4µg of total RNA using the Illumina Truseq® Stranded mRNA Sample Preparation kit according to the manufacturer's protocol (Illumina; RS-122-2101). Briefly, mRNAs were purified and fragmented before first strand synthesis with random hexamers, then the second strand synthesis was performed and adaptors were ligated to the products before PCR amplification of the samples as previously recommended to minimize bias. Indexed libraries were normalized and pooled two by two for random multiplexing. Libraries were subjected to a 75 bp paired-end sequencing on an Illumina GAIIx, according to the manufacturer's protocol. Two samples were pooled on a flowcell lane. Raw image files were processed using the Illumina Real Time Analysis (RTA1.9) software (Illumina). Demultiplexing was performed using CASAVA 1.8.2 (Illumina). On average, 38 millions of reads were generated per sample corresponding to 2.9 Gb per sample. 94% of bases were read with a Qscore above 30.