Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA016458

Local Sample ID:	CM_CdPool_2
Subject ID:	SU000386
Subject Type:	Zebrafish
Subject Species:	Danio rerio
Taxonomy ID:	7955
Species Group:	Fish

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Sample Preparation:

Sampleprep ID:	SP000393
Sampleprep Summary:	Pools with 40 larvae each were washed and snap frozen before being shipped to the NIH RTI-RCMRC on dry ice and immediately stored at -80 °C after being logged in for metabolomics analysis. A total of 30 study samples were lyophilized overnight for sample preparation. Each sample was mixed with 750 μl of methanol, vortexed, and centrifuged. 300 μl of the supernatant were used for the study samples, phenotypic pools were created using 350 μl of each phenotype and a total study pool was created by mixing 35 μl from each sample. Three aliquots were created from each pool for a total of 15 pooled samples. All samples were dried in vacuum overnight. Each sample was reconstituted with 250 μl of 0.5 mM phosphate buffer (pH 7.5) and 25 μl of d-DSS/Chenomx as an internal standard. The tubes were vortexed for 2 min on a multi-tube vortexer and centrifuged at 16,000 rcf for 10 min. A 200 µl aliquot of the supernatant was transferred into pre-labeled 3 mm (4”) NMR tubes for data acquisition on a 700 MHz spectrometer. 1H NMR spectra of zebrafish extraction samples were acquired on a Bruker 700 MHz NMR spectrometer (located at David H. Murdock Research Institute, Kannapolis, NC, USA) using a 5 mm cryogenically cooled ATMA inverse probe and ambient temperature of 25 ℃. A 1D NOESY presaturation pulse sequence (noesypr1d, [recycle delay (RD)-90°-t1-90°-tm-90°-acquire free induction decay (FID) was used for data acquisition. For each sample 32 transients were collected into 65k data points using a spectral width of 17.17 ppm, 2 s relaxation delay, 100 ms mixing time, and an acquisition time of 3.9 s per FID. The water resonance was suppressed using resonance irradiation during the relaxation delay and mixing time. NMR spectra were processed using TopSpin 3.2 software (Bruker-Biospin, Germany). Spectra were zero filled, and Fourier transformed after exponential multiplication with line broadening factor of 0.5. Phase and baseline of the spectra were manually corrected for each spectrum. Spectra were referenced internally to the DSS-d6 signal. The quality of each NMR spectrum was assessed for the level of noise and alignment of identified markers. Spectra were assessed for missing data and underwent quality checks.

Sampleprep ID:

SP000393

Sampleprep Summary:

Pools with 40 larvae each were washed and snap frozen before being shipped to the NIH RTI-RCMRC on dry ice and immediately stored at -80 °C after being logged in for metabolomics analysis. A total of 30 study samples were lyophilized overnight for sample preparation. Each sample was mixed with 750 μl of methanol, vortexed, and centrifuged. 300 μl of the supernatant were used for the study samples, phenotypic pools were created using 350 μl of each phenotype and a total study pool was created by mixing 35 μl from each sample. Three aliquots were created from each pool for a total of 15 pooled samples. All samples were dried in vacuum overnight. Each sample was reconstituted with 250 μl of 0.5 mM phosphate buffer (pH 7.5) and 25 μl of d-DSS/Chenomx as an internal standard. The tubes were vortexed for 2 min on a multi-tube vortexer and centrifuged at 16,000 rcf for 10 min. A 200 µl aliquot of the supernatant was transferred into pre-labeled 3 mm (4”) NMR tubes for data acquisition on a 700 MHz spectrometer. 1H NMR spectra of zebrafish extraction samples were acquired on a Bruker 700 MHz NMR spectrometer (located at David H. Murdock Research Institute, Kannapolis, NC, USA) using a 5 mm cryogenically cooled ATMA inverse probe and ambient temperature of 25 ℃. A 1D NOESY presaturation pulse sequence (noesypr1d, [recycle delay (RD)-90°-t1-90°-tm-90°-acquire free induction decay (FID) was used for data acquisition. For each sample 32 transients were collected into 65k data points using a spectral width of 17.17 ppm, 2 s relaxation delay, 100 ms mixing time, and an acquisition time of 3.9 s per FID. The water resonance was suppressed using resonance irradiation during the relaxation delay and mixing time. NMR spectra were processed using TopSpin 3.2 software (Bruker-Biospin, Germany). Spectra were zero filled, and Fourier transformed after exponential multiplication with line broadening factor of 0.5. Phase and baseline of the spectra were manually corrected for each spectrum. Spectra were referenced internally to the DSS-d6 signal. The quality of each NMR spectrum was assessed for the level of noise and alignment of identified markers. Spectra were assessed for missing data and underwent quality checks.