Return to study ST001921 main page
MB Sample ID: SA177912
Local Sample ID: | SARS_CoV_2_3 |
Subject ID: | SU001999 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001999 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
SARS_CoV_2_3 | SA177912 | FL022028 | SARS_CoV_2 | Treatment |
Collection:
Collection ID: | CO001992 |
Collection Summary: | hPSC-AOs were collected into 150 mM ammonium acetate. After centrifugation, cell pellets were collected for metabolite profiling. Metabolite extraction was performed with 200 μl of 80% methanol (20% water) with internal standards (U13C succinate, and U13C citrate, and heptadecanoate). |
Sample Type: | Lung organoids |
Treatment:
Treatment ID: | TR002011 |
Treatment Summary: | SARS-CoV-2 variants, including USA-WA1/2020 and B.1.351, were obtained from the World Reference Center for Emerging Viruses and Arboviruses located at the University of Texas, Medical Branch via the CDC. SARS-CoV-2 was propagated in Vero E6 cells (ATCC) in EMEM supplemented with 10% FCS, 1 mM Sodium Pyruvate and 10 mM HEPES. hPSC-AOs were fragmented into small cell clusters and plated on 10% matrigel-coated plates. The infection was performed in the culture media at the indicated MOIs at 37°C. For pre-infection treatment experiments, hPSC-AOs were pretreated with DMSO (control), 10 µM GW6471 for 4 hours prior to infection. HIF1a knockdown in hPSC-AOs Two shRNAs against HIF1α and one scrambled negative control non-effective shRNA in lentiviral GFP vectors were purchased from OriGene company. The shRNA sequences are shown in Table S3. To generate lentivirus particles, shRNA vectors and lentivirus packaging vectors were co-transfected into 293T cells. The day 15 lung progenitor cells were infected with the collected lentivirus (MOI=0.5) with 8 μg/ml polybrene. To increase the infection efficiency, the cells were centrifuged at 2300 rpm for 1 hour at 30℃. At 24 hpi, the cells were selected with 1 μg/ml puromycin for an additional 72 hours. After selection, the cells were dissociated into single cells and seeded into 24 well plates at 300-400 cells/μl for 3D organoid differentiation. |
Sample Preparation:
Sampleprep ID: | SP002005 |
Sampleprep Summary: | In brief, the samples were methoximized with 50 µl of methoxyamine hydrochloride (MOA, 15 mg/mL in pyridine) at 30°C for 90 minutes. The silylation step was done with 50 µl of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, containing 1% TMCS, Supelco, PA, USA) at 70 °C for 60 minutes. After the samples cooled to room temperature, they were analyzed by GC-TOF/MS. Separation was performed with a 30-meter DB-5MS column coupled with 10-meter guard column. Helium was used as carrier gas at a flow of 1 ml/min. The oven program is as the following: started at 60 °C for 1 minute, 10°C /min to 320 minutes and kept for 3 minutes. |
Combined analysis:
Analysis ID | AN003122 |
---|---|
Analysis type | MS |
Chromatography type | GC |
Chromatography system | Agilent 7890A |
Column | Agilent DB5-MS (30m) |
MS Type | EI |
MS instrument type | GC-TOF |
MS instrument name | Unspecified |
Ion Mode | POSITIVE |
Units | Area |
Chromatography:
Chromatography ID: | CH002307 |
Chromatography Summary: | Separation was performed with a 30-meter DB-5MS column coupled with 10-meter guard column. Helium was used as carrier gas at a flow of 1 ml/min. The oven program is as the following: started at 60 °C for 1 minute, 10°C /min to 320 minutes and kept for 3 minutes. |
Instrument Name: | Agilent 7890A |
Column Name: | Agilent DB5-MS (30m) |
Chromatography Type: | GC |
MS:
MS ID: | MS002903 |
Analysis ID: | AN003122 |
Instrument Name: | Unspecified |
Instrument Type: | GC-TOF |
MS Type: | EI |
MS Comments: | The transfer line temperature was set to 250 °C. The data was acquired with MassLynx software (Waters). The data was collected with the scan time of 0.3 s and interscan delay of 0.1 s. The mass range was set to 35 to 650 for EI. Raw data files (.raw) generated from GC-TOF/MS were analyzed in Refiner MS (Genedata Expressionist, Basel, Switzerland) software. The output from Refiner MS contains retention time, quantification mass, and peak volume for each metabolite. |
Ion Mode: | POSITIVE |