Transcriptome Analysis of Nitrate-Deprived Diatom

The experiment was carried out using a newly established clonal strain of C. socialis, APC12, genotyped by sequencing the LSU rDNA region ([21 ]). A non-axenic stock has been maintained in a culture chamber at 18 ± 2 °C, under sinusoidal illumination (12L:12D h photoperiod, ~ 90 μmol photons·m⁻²· s⁻¹ daily average) in control medium made with artificial seawater at a salinity of 36 (Sea salts, Sigma-Aldrich; [21 ]) and with the following concentration of inorganic nutrients: 580 μM of NaNO₃, 300 μM of Na₂SiO₃ and 29 μM of NaH₂PO₄. An exponentially growing culture was used to inoculate, at an initial cell density of ~ 3 × 10³ cells·mL⁻¹, three 5 L glass flasks filled with 3 L of control medium and three flasks filled with low nitrate medium (23 μM of NaNO₃, 300 μM Na₂SiO₃, 29 μM NaH₂PO₄). Temperature and light conditions were monitored during the experiment with a HOBO Pendant® Temperature/Light Data Logger. To estimate cell concentration, 4 mL of sample were collected every day, fixed with 1.6% formaldehyde solution, and vegetative cells and spores were enumerated using a Sedgwick-Rafter chamber on a Zeiss Axiophot (ZEISS, Oberkochen, Germany) microscope at 400 × magnification.
Total RNA was extracted from each replicate of the control in mid-exponential growth phase at day 2 (C2) and from the replicates growing in N deplete conditions on three consecutive days: before the formation of spores (T2), when spore formation started (T3), and when they reached > 75% of the whole population (T4) (Fig. 6). A total of ~ 1.2 × 10⁷ cells were harvested from each replicate by filtration onto 1.2 μm pore size filters (RAWP04700 Millipore) and extracted with Trizol™ (Invitrogen) following manufacturer’s instructions. A DNase I (Qiagen) treatment was applied to remove gDNA contamination, and RNA was further purified using RNeasy Plant Mini Kit (Qiagen). All samples were quantified with Qubit® 2.0 Fluorometer (Invitrogen) and quality checked with an Agilent 2100 bioanalyzer (Agilent Technologies, California, USA) and a NanoDrop ND-1000 Spectrophotometer (Nanodrop Tecnologies Inc., Wilmington, USA). Samples were then pooled in equal concentrations of 100 ng·μl⁻¹ for sequencing at the Molecular Service of Stazione Zoologica with an Ion Proton™ sequencer (Life Technologies, Carlsbad, USA) using an Ion P1 sequencing Kit v2, generating single-read sequences. Highly abundant ribosomal RNAs (rRNA) were removed from total RNA by positive polyA + selection. Raw reads coming from each replicate were collected in fastqc format files. One of the T3 replicates was removed from downstream analyses due to a sequencing error during library construction. The resulting raw reads were deposited in the Sequence Read Archive (SRA) partition at NCBI with the accession number PRJNA826817.Fig. 6

Schematic representation of the bioinformatic pipeline used in this study