Further Details
Culture experiments carried out with Gephyrocapsa huxleyi CCMP2758 cells grown in a 3L batches in 5L Nalgene bottles. Cultures were kept in a climate controlled room at 7C, they were continuously stirred and air was constantly provided using an aquarium air pump through a 0.2 �m pore size filter (Sartorius, G�ttingen, Germany). Light intensity: 80-115 �E. Cell growth was monitored daily using flow cytometry. Upon reaching a cell density of 1933019.398 cells/mL. Cells were harvested by filtering 50 mL of culture over GF/F filters as described above and immediately stored at -80C until further processing.
strain
CCMP2758
amount or size of sample collected
0.05
sample storage temperature
-80
organism
Emiliania huxleyi
collection date
2019-10-26
water temperature
7
scientific_name
Emiliania huxleyi
collected_by
Ruth Perez Gallego
sample collection method
Filtration over GF/F filter
culture_collection
National Center for Marine Algae and Microbiota (NCMA)
sample storage conditions
Frozen
nucleic acid extraction
RNA was extracted from one quarter of each of glass fiber GF/F filter using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer�s instructions with the following modifications: after addition of 1 mL lysis buffer supplemented with 10 ul beta-mercaptoethanol to the cut filters, 50ul of Ambion Plant RNA isolation Aid (Ambion, Lithuania) were added to optimize RNA extraction. Filters were bead beated using a mixture of 0.2 mL 0.5 mm diameter and 0.2 mL 0.1 mm diameter glass beads (BioSpec Products, Bartlesville, OK, USA) twice for 30 seconds at a speed of 4 m s-1 with a 30 second dwell using a bead mill homogenizer (Bead Ruptor Elite, Omni International, Kennesaw, GA, USA). After centrifugation of the extracts, 2 ul of SUPERase In RNase Inhibitor 20 U ul-1 (Invitrogen, Carlsbad, CA, USA) was added to the cleared lysate. On-column DNA digestion during RNA extraction was performed using On-Column DNAse I Digestion Set (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer�s instructions.  Additionally, RNA eluates were treated twice with TURBO DNA-free kit to remove possible DNA contamination. PCR was performed on RNA extracts to confirm the absence of DNA.  20 U ul-1 (Invitrogen, Carlsbad, CA, USA) was added to the cleared lysate. On-column DNA digestion during RNA extraction was performed using On-Column DNAse I Digestion Set (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer�s instructions.  Additionally, RNA eluates were treated twice with TURBO DNA-free kit to remove possible DNA contamination. PCR was performed on RNA extracts to confirm the absence of DNA. The quality and integrity of the RNA extracts was assessed using an Agilent RNA 6000 Nano Kit on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
geographic location (country and/or sea)
missing: lab stock