Chlorella sorokiniana
Protocols: C. sorokiniana cells (triplicates of irradiated and control treatments) were collected by centrifugation at 5000 g/5 min, washed 3× with ice-cold deionized water, snap-frozen in liquid N2, and stored at -80°C until further analysis. RNA extraction was performed by the addition of 1 mL TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA USA), which was added to each sample and left to incubate at room temperature for 5 min. Chloroform (200 µL) was added to each sample for further extraction. Samples were briefly vortexed and left to stand for additional 5 min. To isolate RNA from DNA, proteins and other cell debris, the samples were centrifuged for 15 min at 12000 g and 4°C. The upper layer was then aliquoted, taking care not to disturb the lower layers. To precipitate the RNA, 500 µL of isopropanol was added to each sample and left for 15 min at room temperature. To collect the precipitated RNA, samples were centrifuged for 10 min at 12000 g and 4°C and the supernatant was removed. The RNA pellet was washed two times with 1 mL ethanol (75%) and centrifugation at 12000 g/5 min/4°C. Samples were left on ice for 30 min to allow any remaining ethanol to evaporate. The RNA pellet was then resuspended in 50 µL sterile deionized H2O. Chlorella sorokiniana strain CCAP 211/8K (alternative designation UTEX 1230) was obtained from the Culture Collection of Algae and Protozoa (CCAP), UK. C. sorokiniana was grown in 35 mL aliquots of 3N-BBM+V medium in 100 mL Erlenmeyer flasks that were placed on an orbital shaker (120 rpm) in a growth cabinet with temperature maintained at 22°C, and a continuous photon flux of 120 μmol m2/s. The medium was inoculated with 0.5 × 106 cells/mL. Microalgae were treated by a low dose of X-ray irradiation compared to non-irradiated control cells. Microalgae were irradiated at 20 days after inoculation, in the early stationary phase then RNA-Seq analysis was performed 24 hours after irradiation. Samples (35 mL) were placed in a Petri dish and exposed to a single continuous dose of X-rays using a CellRad system (FaxitronBioptics LLC, Tucson, AZ, USA), with the following settings: tube power, 750 W; filtration, 1.6 mm Be and 0.5 mm Al; energy, 120 kV, and using an irradiation dose of 5 Gy at a dose rate of 0.5 Gy/min, measured by using the built-in ion chamber dosimeter. Samples were rotated during irradiation. Irradiated cultures were placed back into 100 mL Erlenmeyer flasks and grown under the same growth conditions as before until cells were harvested for RNA extraction after 24 hours. Treated samples and controls (non-irradiated samples) were grown in the same batch. All experiments were performed in biological triplicates. Chlorella sorokiniana strain CCAP 211/8K (alternative designation UTEX 1230) was obtained from the Culture Collection of Algae and Protozoa (CCAP), UK. C. sorokiniana was grown in 35 mL aliquots of 3N-BBM+V medium in 100 mL Erlenmeyer flasks that were placed on an orbital shaker (120 rpm) in a growth cabinet with temperature maintained at 22°C, and a continuous photon flux of 120 μmol m2/s. The medium was inoculated with 0.5 × 106 cells/mL. Microalgae were treated by a low dose of X-ray irradiation compared to non-irradiated control cells. Microalgae were irradiated at 20 days after inoculation, in the early stationary phase then RNA-Seq analysis was performed 24 hours after irradiation. Samples (35 mL) were placed in a Petri dish and exposed to a single continuous dose of X-rays using a CellRad system (FaxitronBioptics LLC, Tucson, AZ, USA), with the following settings: tube power, 750 W; filtration, 1.6 mm Be and 0.5 mm Al; energy, 120 kV, and using an irradiation dose of 5 Gy at a dose rate of 0.5 Gy/min, measured by using the built-in ion chamber dosimeter. Samples were rotated during irradiation. Irradiated cultures were placed back into 100 mL Erlenmeyer flasks and grown under the same growth conditions as before until cells were harvested for RNA extraction after 24 hours. Treated samples and controls (non-irradiated samples) were grown in the same batch. All experiments were performed in biological triplicates. C. sorokiniana cells (triplicates of irradiated and control treatments) were collected by centrifugation at 5000 g/5 min, washed 3× with ice-cold deionized water, snap-frozen in liquid N2, and stored at -80°C until further analysis. RNA extraction was performed by the addition of 1 mL TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA USA), which was added to each sample and left to incubate at room temperature for 5 min. Chloroform (200 µL) was added to each sample for further extraction. Samples were briefly vortexed and left to stand for additional 5 min. To isolate RNA from DNA, proteins and other cell debris, the samples were centrifuged for 15 min at 12000 g and 4°C. The upper layer was then aliquoted, taking care not to disturb the lower layers. To precipitate the RNA, 500 µL of isopropanol was added to each sample and left for 15 min at room temperature. To collect the precipitated RNA, samples were centrifuged for 10 min at 12000 g and 4°C and the supernatant was removed. The RNA pellet was washed two times with 1 mL ethanol (75%) and centrifugation at 12000 g/5 min/4°C. Samples were left on ice for 30 min to allow any remaining ethanol to evaporate. The RNA pellet was then resuspended in 50 µL sterile deionized H2O. TruSeq Stranded mRNA
isolate
not applicable
ENA-FIRST-PUBLIC
2023-09-13T00:24:03Z
cultivar
CCAP 211/8K
organism
Chlorella sorokiniana
organism
Chlorella sorokiniana
ENA-LAST-UPDATE
2023-09-13T00:24:03Z
scientific_name
Chlorella sorokiniana
organism part
whole organism
age
21
genotype
wild type genotype
ENA-FIRST-PUBLIC
2023-09-13
ENA-LAST-UPDATE
2023-09-13