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MB Sample ID: SA241941
| Local Sample ID: | F2-27-09 |
| Subject ID: | SU002502 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002502 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| F2-27-09 | SA241941 | FL030327 | K562 Oe-IFITM3 | Group |
Collection:
| Collection ID: | CO002495 |
| Collection Summary: | The human embryonic kidney 293T (HEK293T) and K562 cells were maintained in Iscove's modified Dulbecco’s medium (IMDM; Sigma), human THP1 cells were maintained in Roswell Park Memorial Institute medium (RPMI; Lonza). All media were supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 IU/ml), streptomycin (100 µg/ml) and 2% glutamine. A549 and Vero E6 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% Non-essential amino acid (NEAA, Gibco), 1% Sodium Pyruvate (Gibco). Calu3 were cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 20% fetal bovine serum (FBS; Gibco), 1% Non-essential amino acid (NEAA, Gibco), 1% Sodium Pyruvate (Gibco) and 2% GlutaMAX (Gibco). All cells were maintained in a 5% CO2 humidified atmosphere at 37°C. A549-hACE2 cell lines were generated by lentiviral transduction. Lentiviral vectors were produced following a standard procedure based on calcium phosphate co-transfection with 3rd generation helper and transfer plasmids. Primary cells Human CD34+ HSPC were isolated through positive magnetic bead selection according to manufacturer’s instructions (Milteney) from umbilical cord blood or from mononuclear cells collected upon informed consent from healthy volunteers according to the Institutional Ethical Committee approved protocol (TIGET01). Otherwise, CB, bone marrow (BM) or G-CSF mPB-CD34+ cells were directly purchased from Lonza or Hemacare. All cells were maintained in a 5% CO2 humidified atmosphere at 37°C. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002514 |
| Treatment Summary: | Transduction All cells were transduced at the indicated multiplicity of infection (MOI) as calculated by titration of vector stocks on 293T cells and expressed as transducing units (TU)/293T cell. Cell lines were transduced with LV for 16 hours, washed and kept in culture until reading transduction efficiency by flow cytometry. Human CB-derived HSPC were cultured in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), 100 ng/ml recombinant human stem cell factor (rhSCF), 20 ng/ml recombinant human thrombopoietin (rhTPO), 100 ng/ml recombinant human Flt3 ligand (rhFlt3), and 20 ng/ml recombinant human IL6 (rhIL6) (all from Peprotech) 16 to 24 hours prior to transduction. HSPC were then transduced at a concentration of 1×106 cells per milliliter with VSV-gp-pseudotyped SINLV for 16 hours at the indicated multiplicity of infection (MOI) in the same medium. G-CSF mobilized peripheral blood CD34+ cells were maintained in culture in CellGro medium (Cell Genix) containing a cocktail of cytokines: 60 ng/ml IL-3, 100 ng/ml TPO, 300 ng/ml SCF, and 300 ng/ml FLT-3L (all from Cell Peprotech). Transductions were performed with the indicated dose of vectors for 16 hours in the same cytokine-containing medium. For single hit reporter LV transductions cells were washed and maintained in serum-free medium supplemented with cytokines as above until the reading of the percentage of positive cells by FACS. To perform OE, KD and KO experiments cells were transduced with the respective LV and then challenged with a second transduction with or without drugs. Except for primary cells, KD and KO cells were selected with Puromycin before the second hit of transduction. |
Sample Preparation:
| Sampleprep ID: | SP002508 |
| Sampleprep Summary: | Lipids were extracted from frozen cell pellets using LC-MS grade cold methanol at a concentration of 2e6 cells/mL. Samples were briefly vortexed to mix then incubated for 30 min at -20C. Supernatants were clarified by centrifugation (10 min, 18,213 g, 4C) then 25 uL aliquots were transferred to autosampler vials. |
Combined analysis:
| Analysis ID | AN003933 | AN003934 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002912 |
| Chromatography Summary: | 17 minute negative |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-1 min 25% B and 0.3 ml/min; 1-2 min 25-50% B and 0.3 mL/min; 2–8 min 50–90% B and 0.3 mL/min; 8–10 min 90–99% B and 0.3 mL/min; 10–14 min hold at 99% B and 0.3 mL/min; 14–14.1 min 99–25% B and 0.3 mL/min; 14.1–16.9 min hold at 25% B and 0.4 mL/min; 16.9–17 min hold at 25% B and resume flow of 0.3 mL/min |
| Flow Rate: | varies, see solvent gradient |
| Solvent A: | 25% acetonitrile; 5 mM ammonium acetate |
| Solvent B: | 50% isopropanol/45% acetonitrile/5% water; 5 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002913 |
| Chromatography Summary: | 17 minute positive |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0–10 min 30–100% B and 0.325 mL/min; 10–12 min 100% B and 0.325 mL/min; 12–12.5 min 100–30% B and 0.4 mL/min; 12.5–14.9 min 30% B and 0.4 mL/min; 14.9–15 min 30% B and 0.325 mL/min. |
| Flow Rate: | varies, see solvent gradient |
| Solvent A: | 25% acetonitrile/75% water; 5 mM ammonium acetate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 5 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003671 |
| Analysis ID: | AN003933 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | the mass spectrometer operated in top 10 data dependent MS2 mode with NCE of 30 eV (HCD) and dynamic exclusion of 10 s. MS1 scans were acquired at a resolution of 70,000 across the m/z range of 125-1500. MS2 scans were acquired at a resolution of 17,500. Sheath and auxiliary gases (both N2) were set at 45 and 15 (unitless), respectively. Instrument stability and quality control were assessed via injection of technical replicates every 6 runs. Untargeted lipid results were obtained using LipidSearch (Thermo) with precursor ion tolerance set to 5 ppm and product ion tolerance at 8 ppm. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003672 |
| Analysis ID: | AN003934 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | the mass spectrometer operated in top 10 data dependent MS2 mode with NCE of 30 eV (HCD) and dynamic exclusion of 10 s. MS1 scans were acquired at a resolution of 70,000 across the m/z range of 125-1500. MS2 scans were acquired at a resolution of 17,500. Sheath and auxiliary gases (both N2) were set at 45 and 15 (unitless), respectively. Instrument stability and quality control were assessed via injection of technical replicates every 6 runs. Untargeted lipid results were obtained using LipidSearch (Thermo) with precursor ion tolerance set to 5 ppm and product ion tolerance at 8 ppm. |
| Ion Mode: | POSITIVE |
