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MB Sample ID: SA090776
Local Sample ID: | SC_20180803_RPLCp_DDA2_Sup_QC_06 |
Subject ID: | SU001312 |
Subject Type: | Bacteria |
Subject Species: | Escherichia coli |
Taxonomy ID: | 562 |
Gender: | Not applicable |
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Sample Preparation:
Sampleprep ID: | SP001320 |
Sampleprep Summary: | Global untargeted metabolomic analyses were performed on the supernatant-fraction of ASB and cystitis pools. Aliquots of each pool (200µL) were added to individual Eppendorf tubes containing 200µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0)) (LC-MS grade). Labeled creatinine-D3 and lysine-D4 were added to each sample to assess the metabolite extraction (sample preparation) step. Samples were first subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samples were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing supernatant was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. The pellet-fraction of each sample pool prepared as described above was lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade), followed by sonication in an ice bath for 10 min. Sample volume for each pool was adjusted such that all samples have the same cell number in each vial. Samples were first subjected to protein precipitation by addition of 1000µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samplwere were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing extract was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. |