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MB Sample ID: SA004291
| Local Sample ID: | SBEP_STM_LPM20_3 |
| Subject ID: | SU000104 |
| Subject Type: | Bacterial cells |
| Subject Species: | Salmonella typhimurium |
| Taxonomy ID: | 90371 |
| Genotype Strain: | CDC 6516-60 |
| Cell Biosource Or Supplier: | ATCC 14028 |
| Species Group: | Microorganism |
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Sample Preparation:
| Sampleprep ID: | SP000100 |
| Sampleprep Summary: | Cell pellets resuspended in ammonium bicarbonate, lysed via bead beating, extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v) |
| Sampleprep Protocol Comments: | Cell pellets were stored at -80°C prior to thawing and were subsequently resuspended in an appropriate volume of 100 mM ammonium bicarbonate according to their wet weight to compensate for any differences in cell numbers. The cells were lysed by bead-beating, and the lysates were transferred into new tubes. Subsequently, 100 µL aliquots of cell lysates were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA). All samples were kept at -80°C prior to chemical derivatization for GC-MS analysis. Dried metabolite extracts were briefly dried again to remove any residual water prior to chemical derivatization. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with generous shaking for 90 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min. The samples were allowed to cool to room temperature and were then analyzed by GC-MS in random order. For technical replicates, each of the derivatized samples was split into two different vials and analyzed separately. |
| Processing Method: | Lysed via bead-beating |
| Extraction Method: | four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA) |
| Extract Concentration Dilution: | chloroform/methanol (2:1, v/v) |
| Extract Enrichment: | dried in vacuo |
| Extract Storage: | dried in vacuo, stored at -80° C |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
