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MB Sample ID: SA176885
Local Sample ID: | Ding_D-6_024 |
Subject ID: | SU001987 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | ATF3(+/+) vs ATF3(-/-) |
Cell Strain Details: | HCT116 cells engineered to knockout ATF3 expression via rAAV-mediated homologous recombination |
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Subject:
Subject ID: | SU001987 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | ATF3(+/+) vs ATF3(-/-) |
Cell Strain Details: | HCT116 cells engineered to knockout ATF3 expression via rAAV-mediated homologous recombination |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Ding_D-6_024 | SA176885 | FL021932 | KO-serine(-) | Genotype_Media supplement |
Collection:
Collection ID: | CO001980 |
Collection Summary: | Cells were counted, washed with cold PBS and then flash-frozen in liquid nitrogen |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR001999 |
Treatment Summary: | HCT116 cells (~50% confluence) were washed with PBS, and cultured in HEPES buffered Krebs-Ringer solution supplemented with 25 mM [U-13C]-D-glucose, 10% dialysed FBS, 2×MEM amino acids, 2× Vitamin Solution and 4 mM L-glutamine, with or without 0.4 mM serine and glycine, for 24 h. Cells were washed with PBS and metabolites extracted using 1 mL of acetonitrile:isopropanol:water (3:3:2, v/v/v) mixture. |
Sample Preparation:
Sampleprep ID: | SP001993 |
Sampleprep Summary: | The extraction solvent was degassed and pre-chilled at −20°C. Samples were homogenized using Geno/Grinder 2010 (SPEX SamplePrep) at 1500 rpm for 30s, then shaken at 4°C for 5 min and centrifuged for 2 minutes at 14,000 rcf. 450 μL supernatant was transferred to a new tube and dried down using Centrivap cold trap concentrator (Labconco). 10 μL of methoxyamine hydrochloride in pyridine (40 mg/mL) was added to dried sample and shaken at 30°C for 1.5 hours for methoximation. 90 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, Sigma-Aldrich) was used for tertbutylsilylation. C8–C30 fatty acid methyl esters (FAMEs) were added to MTBSTFA as internal standards for retention time correction. Samples were shaken at 37°C for 30 min for TMS or shaken at 80°C for 30 min for TBS and then ready for injection. |
Combined analysis:
Analysis ID | AN003106 |
---|---|
Analysis type | MS |
Chromatography type | GC |
Chromatography system | Agilent 7890A |
Column | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
MS Type | EI |
MS instrument type | GC-TOF |
MS instrument name | Agilent 7890A |
Ion Mode | UNSPECIFIED |
Units | enrichment percentage |
Chromatography:
Chromatography ID: | CH002293 |
Chromatography Summary: | Gas chromatography-Quadrupole-time of flight mass spectrometry (GC-Q-TOF MS) was used for Stable Isotope enrichment. Rtx-5Sil MS column (30m length, 0.25 mm i.d, 0.25 μM 95% dimethyl 5% diphenyl polysiloxane film) with an additional 10 m guard column was installed on Agilent 7890 GC (Agilent Technologies). 99.9999% pure Helium gas was used as a mobile phase with a flow rate of 1mL/min. GC temperature was held at 50°C for 1 min, ramped at 20°C/min to 330°C and then held for 5 min. Electron ionization at -70eV was employed on a Agilent 7250 Quadrupole time of flight mass spectrometer (Agilent Technologies) with ion source temperature at 250°C and detector voltage at 1850V. Mass spectra were acquired at an acquisition rate of 5 spectra/s, with a scan range of 85-900 Da. |
Instrument Name: | Agilent 7890A |
Column Name: | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
Chromatography Type: | GC |
MS:
MS ID: | MS002888 |
Analysis ID: | AN003106 |
Instrument Name: | Agilent 7890A |
Instrument Type: | GC-TOF |
MS Type: | EI |
MS Comments: | Agilent MassHunter Quantitative Analysis software B.10.00 (Agilent Technologies) was used for post data processing. |
Ion Mode: | UNSPECIFIED |