Further Details
CCMP1742 cells grown in a 8L batches in 10L Nalgene bottles. Cultures were kept in a climate controlled room at 20C, 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: 105-125 �E
strain
CCMP1742
amount or size of sample collected
6
sample storage temperature
-80
organism
Emiliania huxleyi
collection date
2019
water temperature
20
scientific_name
Emiliania huxleyi
collected_by
Ruth Perez Gallego
sample collection method
Continuous centrifugation. Biomass was harvested by pumping the culture into a continuous centrifuge (Heraeus Contrifuge Stratos, Thermo Electron LED, Osterode, Germany) set to 5000 using a peristaltic pump (Masterflex L/S easy load II, Masterflex, Vernon Hills, IL, USA) at a flow rate of 200 mL min-1. The rotor was cleaned using one liter of ultrapure water, 100 mL hypochlorite 1% solution followed by one liter of ultrapure water and one liter of autoclaved seawater prior to culture centrifugation. After c.a. 6 liters of CCMP1742 had been centrifuged the sedimented cultures were washed by pumping an additional one liter of sterilized filtered seawater in the continuous centrifuge. Upon disassembling the rotor, the remaining supernatant was removed, and the pellet was collected using a pipette with a cut-off tip. Cells were further concentrated at room temperature on a bench top centrifuge (Eppendorf Centrifuge 5810R) set at 4000 rpm for 20 minutes. Pellets were then immediately frozen using liquid nitrogen and stored at -80 ?C until further processing.
culture_collection
National Center for Marine Algae and Microbiota (NCMA)
sample storage conditions
Frozen
nucleic acid extraction
Extraction of unsheared and high molecular weight DNA carried out using an amount of concentrated pellet equivalent to approximately 500 mL of culture was used. Each pellet was resuspended in a solution containing 800 ?l of CTAB � Lysis buffer Biochemica (PanReac AppliChem, Darmstadt, Germany), 0.8 mg mL-1 lysozyme (Thermo Scientific�, Waltham, MA), 5 ?g mL-1 RNAse A (Qiagen, Mat. No. 1007885) and incubated at 37 ?C for 60 min, whilst gently shaking the tube every 10 min. To inactivate potential DNAses present in the reagent, RNase A solution (Qiagen, Valencia, CA, USA) was incubated at 50 ?C for 10 min and then added to the lysate-CTAB mix. Afterwards a solution containing RNA grade-Proteinase K (Invitrogen, Carlsbad, CA, USA) and SDS (Ambion, Invitrogen, Carlsbad, CA, USA) were added to the mixture at a final concentration of 0.2 mg mL-1 and 0.5% respectively, and incubated at 60 ?C for 60 min, whilst gently shaking the tube every 10 min. After incubation, 1 volume of Phenol:Chloroform:Isoamyl alcohol (25:24:1) was added and mixed by gently inverting the tube. Samples were then centrifuged at room temperature for 15 min at 1500 g, the top aqueous phase was recovered in a new Eppendorf tube and mixed with one volume of Chloroform:Isoamyl alcohol (24:1) by gently inverting the tube. After centrifugation at 1500 g for 5 min, the aqueous phase was recovered, mixed with 1 volume of Chloroform and centrifuged. The DNA present in the recovered aqueous phase was precipitated via overnight incubation at -20 ?C in a solution containing: 1:100 vol of Glycogen solution (5 mg mL-1) (Invitrogen, Vilnius, Lithuania), 1:10 (v/v) 3 M Sodium acetate pH 5.5 and 2.5x (v/v) cold ethanol 100%. After overnight precipitation DNA was pelleted by centrifugation for 20 min at 13 000 rpm and 4 ?C. Supernatant was discarded and the pellet was washed five times as follows: pellet was resuspended with cold ethanol 70%, centrifuged for 5 min at 13 000 rpm (4 ?C) and incubated at 37 ?C until dry. DNA was eluted using 50 ?l Tris-EDTA (10mM tris, 1 mM EDTA at pH 8) (Invitrogen, Vilnius, Lithuania) and incubated at 37 ?C for 30 min. Quality of the high molecular weight DNA extracts was assessed by running a dilution of the extracts on a 1 % agarose gel.
geographic location (country and/or sea)
missing: lab stock