Return to study ST002412 main page
MB Sample ID: SA241925
| Local Sample ID: | C220181610_PKR_Null_2 |
| Subject ID: | SU002501 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Spleen-derived macrophage from WT or Eif2ak2-/- |
| Age Or Age Range: | NA |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002501 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Spleen-derived macrophage from WT or Eif2ak2-/- |
| Age Or Age Range: | NA |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Not applicable |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| C220181610_PKR_Null_2 | SA241925 | FL030323 | PKR knockout | treatment |
Collection:
| Collection ID: | CO002494 |
| Collection Summary: | Exogenous genes were stably expressed and endogenous gene expression suppressed in murine macrophages by transduction with lentiviral constructs. Gene open reading frames were cloned as NheI-BamHI (New England Biolabs) fragments in the plentiCRISPR v2-Blast (Mohan Babu, Addgene plasmid # 83480; http://n2t.net/addgene:83480; RRID: Addgene_83480). Knockdown of endogenous transcripts was achieved with short hairpin RNA cloned into pLKO.1-puro. Lentiviral particles were packaged using a Lenti-X mix (Takara) by transfection of the plasmid components into HEK293FT cells with Lipofectamine 2000 (Invitrogen). The culture supernatant was collected after 48 hours, centrifuged at 10 000 g, then pipetted onto adherent macrophages. Transduced cells were isolated over 2-3 month by increasing selection (from 1 to 20 ug/mL) with puromycin (Thermo Fisher Scientific) and/or blasticidin (InvivoGen), for pLKO-1-puro-shRNA and plentiCRISPR v2-blast, respectively. Recombinant cells were maintained with antibiotics in Dulbecco’s modified Eagle’s Medium (DMEM, GibCo) supplemented with 10% foetal bovine serum (Bovogen) in a humidified 5% CO 2 incubator at 37 °C. Growth on different carbon sources was tested by plating 6x10 3 cells per well of a 24 well plate with glucose free DMEM (ThermoFisher Scientific) supplemented with 10% FBS (not dialysed), 20 ug/mL puromycin and 20 uM of each sugar. After 72 hours the cells were lifted with trypsin and counted by automation (Countess, Invitrogen). |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR002513 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP002507 |
| Sampleprep Summary: | Adherent macrophages were lifted with trypsin, suspended in PBS and 2x10 6 cells were collected by centrifugation. The cell pellet was rinsed in 10 mL ice cold 0.9% NaCl in water at 4 °C pelleted, then resuspended in 200 L of 4 °C extraction solvent (a 1:3:1 ratio of chloroform, methanol and water). The sample was subjected to three freeze-thaw cycles then mixed by vortex for 30 minutes at 4 °C and cell debris was removed by centrifugation at 20,000g for 10 minutes at 4 °C. The cleared supernatant was transferred (180 uL) and frozen at -80°C until LCMS analysis. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003931 | AN003932 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak height | peak height |
Chromatography:
| Chromatography ID: | CH002910 |
| Chromatography Summary: | pHILIC chromatography at pH9 using Orbitrap high resolution accurate mass MS detection with posneg switching |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Pressure: | 60 bar at starting conditions |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Randomization Order: | yes |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002911 |
| Chromatography Summary: | pHILIC chromatography at pH9 using Orbitrap high resolution accurate mass MS detection with posneg switching |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Pressure: | 60 bar at starting conditions |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Randomization Order: | yes |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003669 |
| Analysis ID: | AN003931 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | .raw files containing both pos and neg data (produced by using polarity switching) were converted to a readable format .mzxml, these files were combined and features aligned. Compiled data matrix was imported to IDEOM for metabolite ID and filtering. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 4 kV |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
| MS ID: | MS003670 |
| Analysis ID: | AN003932 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | .raw files containing both pos and neg data (produced by using polarity switching) were converted to a readable format .mzxml, these files were combined and features aligned. Compiled data matrix was imported to IDEOM for metabolite ID and filtering. |
| Ion Mode: | NEGATIVE |
| Capillary Voltage: | 3.5 kV |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
