Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA318304

Local Sample ID:	Sample 15
Subject ID:	SU003046
Subject Type:	Cultured cells
Subject Species:	Symbiodiniaceae

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Subject:

Subject ID:	SU003046
Subject Type:	Cultured cells
Subject Species:	Symbiodiniaceae

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Sample 15	SA318304	FL037875	Symbiodinium microadriaticum	Genotype
Sample 15	SA318304	FL037875	Untreated	Treatment

Collection:

Collection ID:	CO003039
Collection Summary:	Sampled from Symbiodiniaceae cultures in F/2 media
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003055
Treatment Summary:	Symbiodiniaceae cultures Two Symbiodiniaceae cultures were targeted from existing stocks at the University of Technology Sydney, Symbiodinium microadriaticum (ITS2: A1, culture ID: RT61), and Breviolum minutum (ITS2: B1, culture ID: RT2, CCMP2463) as preliminary trials allowed these cultures to be maintained for extended periods in an extracellular bacteria-free state. Each Symbiodiniaceae species was sub-cultured (n = 10 per Symbiodiniaceae species) by adding 10 mL of original cultures in 90 mL of autoclaved and filter sterilised (0.22 µm) artificial seawater (ASW) and F/2 media. Cultures were grown for one month (to achieve a minimum cell density of 106 cells/mL) at 26˚C with an irradiance of 85 ± 15 µmol photons m-2 s-1 (Philips TLD 18W/54 fluorescent tubes, 10 000 K on a 12h:12h light:dark cycle). Before use, cells were centrifuged at 700 × g for 10 mins at 26˚C and rinsed twice with ASW to remove residual media solution. Cells were resuspended in 100 mL ASW + F/2 media in sterile culture flasks. Untreated cultures Untreated Symbiodiniaceae cultures (n = 5) were maintained as above alongside the Ab+Tx treatments (below). Antibiotic treatment (AbTx) Each antibiotic treatment subculture (n = 5 per Symbiodiniaceae species) was provided with TritonX-100 detergent added to a final concentration of 20 µg/mL and placed on a shaker at mid speed for 30 s. All cultures (Ab+Tx treatment and untreated) were immediately centrifuged at 700 × g for 10 mins at 26˚C, and the supernatant discarded. Cells were rinsed in 20 mL ASW and centrifuged at 700 x g for 10 mins at 26˚C. Cells were transferred to sterile culture flasks and resuspended in 9 ml ASW+F/2. An aliquot of 1 mL custom antibiotic mix (Penicillin at 31.25 µg mL-1, Streptomycin and Kanamycin at 50 µg mL-1, and Neomycin, Ciprofloxacin and Ampicillin all at 100 µg mL-1; Ab+Tx) was added to each antibiotic treatment flask (and 1 mL ASW added to each untreated flask), and flasks were replaced in the incubator. After 48 hours, 90 mL ASW + F/2 was added to all cultures. Cultures were allowed to recover for 5 days prior to metabolism quenching.

Treatment ID:

TR003055

Treatment Summary:

Symbiodiniaceae cultures Two Symbiodiniaceae cultures were targeted from existing stocks at the University of Technology Sydney, Symbiodinium microadriaticum (ITS2: A1, culture ID: RT61), and Breviolum minutum (ITS2: B1, culture ID: RT2, CCMP2463) as preliminary trials allowed these cultures to be maintained for extended periods in an extracellular bacteria-free state. Each Symbiodiniaceae species was sub-cultured (n = 10 per Symbiodiniaceae species) by adding 10 mL of original cultures in 90 mL of autoclaved and filter sterilised (0.22 µm) artificial seawater (ASW) and F/2 media. Cultures were grown for one month (to achieve a minimum cell density of 106 cells/mL) at 26˚C with an irradiance of 85 ± 15 µmol photons m-2 s-1 (Philips TLD 18W/54 fluorescent tubes, 10 000 K on a 12h:12h light:dark cycle). Before use, cells were centrifuged at 700 × g for 10 mins at 26˚C and rinsed twice with ASW to remove residual media solution. Cells were resuspended in 100 mL ASW + F/2 media in sterile culture flasks. Untreated cultures Untreated Symbiodiniaceae cultures (n = 5) were maintained as above alongside the Ab+Tx treatments (below). Antibiotic treatment (AbTx) Each antibiotic treatment subculture (n = 5 per Symbiodiniaceae species) was provided with TritonX-100 detergent added to a final concentration of 20 µg/mL and placed on a shaker at mid speed for 30 s. All cultures (Ab+Tx treatment and untreated) were immediately centrifuged at 700 × g for 10 mins at 26˚C, and the supernatant discarded. Cells were rinsed in 20 mL ASW and centrifuged at 700 x g for 10 mins at 26˚C. Cells were transferred to sterile culture flasks and resuspended in 9 ml ASW+F/2. An aliquot of 1 mL custom antibiotic mix (Penicillin at 31.25 µg mL-1, Streptomycin and Kanamycin at 50 µg mL-1, and Neomycin, Ciprofloxacin and Ampicillin all at 100 µg mL-1; Ab+Tx) was added to each antibiotic treatment flask (and 1 mL ASW added to each untreated flask), and flasks were replaced in the incubator. After 48 hours, 90 mL ASW + F/2 was added to all cultures. Cultures were allowed to recover for 5 days prior to metabolism quenching.

Sample Preparation:

Sampleprep ID:	SP003052
Sampleprep Summary:	Seven days after the antibiotic treatment process, 3 × 1 mL aliquots were collected from each culture flask for flow cytometry cell counting of both bacteria and Symbiodiniaceae cells, bacteria community analysis via 16S rRNA amplicon sequencing analysis, and Symbiodiniaceae photophysiological measurements (methods described in more detail below). The remaining Symbiodiniaceae cells were concentrated by centrifugation at 700 × g for 10 mins at 26˚C, the media discarded, and Symbiodiniaceae pellets snap frozen in liquid nitrogen for metabolite profiling.

Sampleprep ID:

SP003052

Sampleprep Summary:

Seven days after the antibiotic treatment process, 3 × 1 mL aliquots were collected from each culture flask for flow cytometry cell counting of both bacteria and Symbiodiniaceae cells, bacteria community analysis via 16S rRNA amplicon sequencing analysis, and Symbiodiniaceae photophysiological measurements (methods described in more detail below). The remaining Symbiodiniaceae cells were concentrated by centrifugation at 700 × g for 10 mins at 26˚C, the media discarded, and Symbiodiniaceae pellets snap frozen in liquid nitrogen for metabolite profiling.

Combined analysis:

Analysis ID	AN004811
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu GC-2010
Column	Agilent DB5-MS (15m x 0.25mm, 0.25um)
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Shimazdu 2030
Ion Mode	POSITIVE
Units	relative abundance

Chromatography:

Chromatography ID:	CH003636
Instrument Name:	Shimadzu GC-2010
Column Name:	Agilent DB5-MS (15m x 0.25mm, 0.25um)
Column Temperature:	100-320
Flow Gradient:	10°C min-1
Flow Rate:	1 mL/min
Solvent A:	helium
Solvent B:	helium
Chromatography Type:	GC

MS:

MS ID:	MS004557
Analysis ID:	AN004811
Instrument Name:	Shimazdu 2030
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	Approximately 520 quantifying MRM targets were collected using Shimadzu Smart Database along with qualifier for each target which covers about 350 endogenous metabolites and multiple 13C labelled internal standards. Both chromatograms and MRMs were evaluated using the Shimadzu GCMS browser and LabSolutions Insight software.
Ion Mode:	POSITIVE