Further Details
Culture experiments carried out with Gephyrocapsa huxleyi CCMP1516 cells grown in 5 L glass Erlenmeyer flasks at 20�C. Cells were grown up to cell densities of ca. 6.5 x 10^5 cells ml-1 , thoroughly mixed, and subsequently transferred into twenty-one 250 ml Erlenmeyer flaks with a wide opening. All flasks were kept at 20�C for 1 day then nine out of twenty-one Erlenmeyer flasks were randomly selected and transferred to a 10�C room with the same light-dark cycle (16:8 h) and level of irradiance (100umol photons m-2 s-1)(T-0; 2.5 h after the dark cycle finished). The change in in-situ growth temperatures of the cultures was monitored using a thermometer within an additional flask containing G. huxleyi. Culture kept at 10C and harvested  2 h after the cold shock by filtering the culture over GF/F filters and immediately stored at -80C until further processing.
strain
CCMP1516
amount or size of sample collected
0.125
sample storage temperature
-80
organism
Emiliania huxleyi
collection date
2017
water temperature
10
scientific_name
Emiliania huxleyi
collected_by
Marc Besseling
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