Cultures: Axenic Thalassiosira pseudonana (CCMP 1335, National Center for Marine Algae and Microbiota, Maine, USA; LSID urn:lsid:marinespecies.org:taxname:148934) batch cultures were grown in enriched artificial seawater (ESAW) medium modified with reduced levels of nitrate (170 μM) to characterize starvation. Before the experiment, the diatoms were acclimated to a constant 20 °C temperature and under a 12: 12 h dark: light diurnal cycle at 300 μmol photons·m−2·s−1 using cool fluorescent lights. The cultures were continuously equilibrated at ambient (420 ppm CO2 by bubbling mixed gasses (air and CO2 at 0.4 L/min) regulated using mass-flow controllers (GFC17, Aalborg) and monitored with a CO2 analyzer (model Q-S151; Qubit Systems) into a 1.5-L glass bioreactor system. The bioreactors were inoculated with 150,000 cells/ml of acclimated, axenic T. pseudonana and grown for 5 days on a 12: 12 h dark: light cycle. The pH was monitored spectrophotometrically (Dickson et al. 2007); the photochemical yield of photosystem II (variable fluorescence/maximum fluorescence Fv/Fm) was measured with an AquaPen AP100; and total dissolved nitrogen was measure using the Nitrite/Nitrate, Colorimetric Test Roche # 11746081001) kit after syringe-filtering (0.2μm) the samples. Samples were taken twice a day, in the middle of dark-time, and the middle of the light-time representing different growth conditions regulating the metabolism based on light and nitrogen on days one and two (Ashworth et al. 2013). On days three and four, samples were only taken during the light time for experiment b. This experiment was repeated to evaluate the recovery of the cells from starvation by amending NO3 (170 uM+/- 5uM) on day 5, after 70 hrs of starvation for T. pseudonana and sampled at T1: 0h, T2: 1h and T3: 3 hr, and T4: 6 hr after adding the supplemental nitrogen. Samples T1, T2, T3, were set on ice in the dark before analysis at when T4 was sampled.
Single cell transcriptomics: we used 10x Genomics Chromium single-cell 3' gene expression protocols to profile samples of 1000-10,000 diatoms (10x Genomics, 2021). We used the standard throughput kits (v3.0, cat. #1000094) for exploration, targeting 10K cells/sample for measurements in a and c, and low throughput (LT) kits (v3.1, cat. #1000325) for a time series targeting 1000 cells/sample in experiment b. Fresh cells were harvested, washed with 1 × Phosphate buffered Saline (PBS) in RNase free water, at pH 7.4 and resuspended at 1 × 106 cells per ml in 1x PBS and 0.10% bovine serum albumin (x). Cellular suspensions were loaded on a Chromium instrument to generate single-cell Gelbead-In-EMulsion (GEM) droplets. Reverse transcription (RT) was performed in a C1000 Touch thermocycler (Biorad, Hercules, CA). After RT, GEMs were harvested and the cDNAs were amplified and cleaned with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA). Indexed sequencing libraries were constructed using the Chromium Single-Cell 3’ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA) and run 150 cycles (26 bp for Read 1 and 124 bp for Read 2).
Experiment Metadata (physiology, gene and cell information):
Experiment
Start_Date 12-4
Light:Dark 12:12
Lights_On 1:00 PM
Lights_(umols/m.s) 1000 umoles m-2s-1
CO2_ppm ~420
scRNAseq_Timepoints 18 hours, 30 hours, 42 hours, 54 hours, 68 hours, 80 hours, 92 hours
scRNAseq_Timepoint_Labels
Notes scRNA, Full ESAW nitrate limited (170uM). Starting innoculate = 150k/m
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