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MB Sample ID: SA333714
| Local Sample ID: | DS1-095-006L- |
| Subject ID: | SU003223 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003223 |
| 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 |
|---|---|---|---|---|
| DS1-095-006L- | SA333714 | FL039622 | cultured reticulocytes | Sample source |
| DS1-095-006L- | SA333714 | FL039622 | 04CF29 | Assay |
Collection:
| Collection ID: | CO003216 |
| Collection Summary: | For flow cytometry, cells were fixed (1% paraformaldehyde, 0.0075% glutaraldehyde) to prevent antibody agglutination, except for assays using GLUT1.RBD. Samples were labelled with primary antibodies in PBSAG (PBS with 1% (w/v) Glucose and 0.5%(w/v) |Bovine Serum Albumin (BSA)) supplemented with extra 1%(w/v) BSA for 30min, in the dark. METAFORA’s GLUT1.RBD was incubated at 37°C and all remaining antibodies incubated at 4°C. Cells were washed 2x PBSAG and, if required, incubated with appropriate secondary antibody under the same conditions as described for primary. Cells were washed 2x with PBSAG and analysed on a Miltenyi MACSQuant 10 flow cytometer. Data was analysed using FlowJo v10.7 (FlowJo). Reticulocytes were identified by gating on the Hoechst-negative population. 4 Cells were sorted using a BD Influx Cell Sorter (BD Biosciences). BEL-A CRISPR edited populations were single cell sorted based on viability (DRAQ7 negativity). Primary GLUT1 KO cultures were sorted on days 6 or 7 of differentiation using DRAQ7 and the eGFP-fused GLUT1.RBD to purify the negative population with a gate based on non-targeting guide control cells. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003232 |
| Treatment Summary: | Filtered reticulocytes (1-2 x105/well) were incubated in decreasing NaCl concentrations (0.9–0%) for 10min at 37°C. Lysis was stopped by adding 4x volume of PBSAG. Live cells, considered as having a normal FSC/SSC profile as defined by the 0.9% NaCl control, were counted by flow cytometry using the MACSQuant10 |
Sample Preparation:
| Sampleprep ID: | SP003229 |
| Sampleprep Summary: | Extraction of metabolites and lipids from red cell pellets was as follows: 36 µL of cold MeOH:MeCN:H2O (5:3:2, v:v:v) was added to all pellets (pellet size pre-determined to be 10 million prior to extraction). Samples were vortexed at 4°C for 30 minutes. Insoluble material was pelleted by centrifugation (18000g, 10 min) and supernatants were isolated for analysis by UHPLC-MS for metabolomics and lipidomics. |
| Sampleprep Protocol Filename: | Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf |
Combined analysis:
| Analysis ID | AN005087 | AN005088 | AN005089 | AN005090 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | area | area | area | area |
Chromatography:
| Chromatography ID: | CH003843 |
| Chromatography Summary: | Metabolomics Positive |
| Methods Filename: | Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0 min - 0.45 ml/min - 5% B, 0.5 min - 0.45ml/min - 5% B, 1.1 min - 0.45ml/min - 95% B, 2.75 min - 0.45ml/min - 95% B, 3 min - 0.45ml/min - 5% B, 5min - 0.45ml/min - 5%B |
| Flow Rate: | 0.45 ml/min |
| Solvent A: | 0.1% Formic Acid in Water |
| Solvent B: | 0.1% Formic Acid in ACN |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003844 |
| Chromatography Summary: | Metabolomics Negative |
| Methods Filename: | Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0 min - 0.45 ml/min - 0% B, 0.5 min - 0.45ml/min - 0% B, 1.1 min - 0.45ml/min - 100% B, 2.75 min - 0.45ml/min - 100% B, 3 min - 0.45ml/min - 0% B, 5min - 0.45ml/min - 0%B |
| Flow Rate: | 0.45 ml/min |
| Solvent A: | 5% ACN 95% Water 1mM Ammonium Acetate |
| Solvent B: | 95% ACN 5% Water 1mM Ammonium Acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003845 |
| Chromatography Summary: | Lipidomics Positive |
| Methods Filename: | Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | 0 min - 0.3ml/min - 30%B, 3 min - 0.3ml/min - 100%B, 4.2min - 0.3ml/min - 100%B, 4.3min - 0.4ml/min - 30%B, 4.9min - 0/4ml/min - 30%B, 5 min - 0.3ml/min 30%B |
| Flow Rate: | 0.3-0.4ml/min |
| Solvent A: | 75:25 H2O:ACN 5mM NH4OAc |
| Solvent B: | 90:10 iPrOH:ACN 5mM NH4OAc |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003846 |
| Chromatography Summary: | Lipidomics Negative |
| Methods Filename: | Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | 0 min - 0.3ml/min - 10%B, 3 min - 0.3ml/min - 95%B, 4.2min - 0.3ml/min - 95%B, 4.3min - 0.45ml/min - 10%B, 4.9min - 0.4ml/min - 10%B, 5 min - 0.3ml/min 10%B |
| Flow Rate: | 0.3-0.45ml/min |
| Solvent A: | 75:25 H2O:ACN 5mM NH4OAc |
| Solvent B: | 90:10 iPrOH:ACN 5mM NH4OAc |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004824 |
| Analysis ID: | AN005087 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | El-Maven used for data analysis. Orbitrap scanning in full MS mode from 65-975 m/z at 60,000 resolution, with 50 Arb sheath gas, 10 Arb auxiliary gas, and 3.4 kV spray voltage. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004825 |
| Analysis ID: | AN005088 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | El-Maven used for data analysis. Orbitrap scanning in full MS mode from 65-975 m/z at 60,000 resolution, with 50 Arb sheath gas, 10 Arb auxiliary gas, and 3 kV spray voltage. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004826 |
| Analysis ID: | AN005089 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Lipidsearch used for data analysis. Orbitrap scanning using data dependent MS2 (top 10) from 125-1500 m/z at 17,500 resolution, with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004827 |
| Analysis ID: | AN005090 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Lipidsearch used for data analysis. Orbitrap scanning using data dependent MS2 (top 10) from 125-1500 m/z at 17,500 resolution, with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. |
| Ion Mode: | NEGATIVE |
