Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA322344

Local Sample ID:	POS_EV1_2
Subject ID:	SU003072
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

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Subject:

Subject ID:	SU003072
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
POS_EV1_2	SA322344	FL038464	Empty vector	Genotype

Collection:

Collection ID:	CO003065
Collection Summary:	70% confluent cultures of control and shKDM2B MDA-MB-231 cells (four biological replicates) were first washed rapidly three times with PBS at room temperature.
Sample Type:	Breast cancer cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR003081
Treatment Summary:	shRNAs in the pLKO-puro lentiviral vector, were packaged in Lenti-X 293T Cells (Takara Bio, Cat. 632180) by transient transfection, in combination with the packaging constructs psPax2 (Addgene, Cat. 12260) and pMD2.G (Addgene, Cat. 12259). Transfections were carried out using the Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Cat. L3000015) and the Opti-MEM Reduced Serum Medium (Fisher Scientific, Cat. 31–985-070), according to the manufacturer’s protocol. The supernatants were collected 48h and 72h after the transfection. MDA-MB-231 and MDA-MB-468 cells were infected with the viral supernatants, in the presence of 8 μg/mL polybrene (Millipore-Sigma, Cat. 107689). Infected cells were selected with puromycin for 48h (Gibco, Cat. A11138) (10 μg/mL).

Treatment ID:

TR003081

Treatment Summary:

shRNAs in the pLKO-puro lentiviral vector, were packaged in Lenti-X 293T Cells (Takara Bio, Cat. 632180) by transient transfection, in combination with the packaging constructs psPax2 (Addgene, Cat. 12260) and pMD2.G (Addgene, Cat. 12259). Transfections were carried out using the Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Cat. L3000015) and the Opti-MEM Reduced Serum Medium (Fisher Scientific, Cat. 31–985-070), according to the manufacturer’s protocol. The supernatants were collected 48h and 72h after the transfection. MDA-MB-231 and MDA-MB-468 cells were infected with the viral supernatants, in the presence of 8 μg/mL polybrene (Millipore-Sigma, Cat. 107689). Infected cells were selected with puromycin for 48h (Gibco, Cat. A11138) (10 μg/mL).

Sample Preparation:

Sampleprep ID:	SP003078
Sampleprep Summary:	1.5–2×106 cells per sample were treated with ice-cold methanol (80% v/v) and they were snap frozen via submergence into liquid Nitrogen for 30 seconds. Subsequently, they were placed on dry ice and allowed to thaw. This step was repeated three times with 10 second vortex-mixing between cycles. At the end, the samples were centrifuged at 11,500 g for 10 min at 4 °C, and the supernatants were collected, lyophilized overnight (~14 h) and stored at −80 °C.

Sampleprep ID:

SP003078

Sampleprep Summary:

1.5–2×106 cells per sample were treated with ice-cold methanol (80% v/v) and they were snap frozen via submergence into liquid Nitrogen for 30 seconds. Subsequently, they were placed on dry ice and allowed to thaw. This step was repeated three times with 10 second vortex-mixing between cycles. At the end, the samples were centrifuged at 11,500 g for 10 min at 4 °C, and the supernatants were collected, lyophilized overnight (~14 h) and stored at −80 °C.

Combined analysis:

Analysis ID	AN004859	AN004860
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Agilent InfityLab Poroshell 120 SB-C18 (100 x 2.1mm, 2.7um)	Agilent InfityLab Poroshell 120 SB-C18 (100 x 2.1mm, 2.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Relative intensity	Relative intensity

Chromatography:

Chromatography ID:	CH003668
Instrument Name:	Agilent 1290 Infinity
Column Name:	Agilent InfityLab Poroshell 120 SB-C18 (100 x 2.1mm, 2.7um)
Column Temperature:	40
Flow Gradient:	0 min, 5%B; 15 min 95%B; 16 min 95%B; 17 min 5%, 25 min 5%B
Flow Rate:	0.2 mL/min
Solvent A:	Water with 0.1 % formic acid
Solvent B:	Methanol with 0.1 % formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004603
Analysis ID:	AN004859
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	ESI configuration included a mass range from 100 to 1,200 m/z, full scan mode at a scan rate of 2 scans per second, 3000V of capillary, 10 L/min of nebulizer gas flow and 300 °C of gas temperature. MS/MS data were collected in data dependent acquisition (DDA) mode with a scan rate of 5 spectra/sec and dynamic exclusion of 30 seconds for precursor ion selection and fragmentation, using 10 to 30 V.
Ion Mode:	POSITIVE

MS ID:	MS004604
Analysis ID:	AN004860
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	ESI configuration included a mass range from 100 to 1,200 m/z, full scan mode at a scan rate of 2 scans per second, 3000V of capillary, 10 L/min of nebulizer gas flow and 300 °C of gas temperature. MS/MS data were collected in data dependent acquisition (DDA) mode with a scan rate of 5 spectra/sec and dynamic exclusion of 30 seconds for precursor ion selection and fragmentation, using 10 to 30 V.
Ion Mode:	NEGATIVE