Return to study ST002289 main page
MB Sample ID: SA219965
| Local Sample ID: | I48.2 |
| Subject ID: | SU002375 |
| Subject Type: | Bacteria |
| Subject Species: | Several mouse commensals |
| Age Or Age Range: | 8-16 hours |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002375 |
| Subject Type: | Bacteria |
| Subject Species: | Several mouse commensals |
| Age Or Age Range: | 8-16 hours |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| I48.2 | SA219965 | FL027399 | I48 | Factor |
| I48.2 | SA219965 | FL027399 | Bacteroides caecimuris | species |
Collection:
| Collection ID: | CO002368 |
| Collection Summary: | Bacteria were grown in 15mL culture tubes in 3mL of modified brain heart infusion broth [37g/L BHI powder, 0.025% Cystiene-HCl.H2O, 0.025% Na2S.9H2O, 1ug/mL Hemin, 0.5ug/mL menadione, 0.025% mucin]. Media was pre-reduced in an anaerobic chamber (Whitley A95 Workstation; 90% N2, 5% H2 & 5% CO2) for 48 hours then bacterial cultures were started from glycerol stocks. 200µL of glycerol stock was added to each culture tube and cultures were incubated for 6-36 hours until early plateau phase. After incubation, cultures were removed from the anaerobic chamber, and 50uL was collected to extract DNA for 16S sequencing to confirm accurate identification of bacteria and absence of contamination. The remaining culture was centrifuged (max. speed, 15 minutes, 4°C) and the supernatant was collected and combined 1:1 with 100% methanol. Samples were then incubated at - 20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4°C), supernatant was recovered and combined 1:4 with 50% methanol to obtain a final dilution of D20. |
| Sample Type: | Bacterial cells |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR002387 |
| Treatment Summary: | Culture conditions and extractions methods described in protocol. |
Sample Preparation:
| Sampleprep ID: | SP002381 |
| Sampleprep Summary: | The remaining culture was centrifuged (max. speed, 15 minutes, 4°C) and the supernatant was collected and combined 1:1 with 100% methanol. Samples were then incubated at - 20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4°C), supernatant was recovered and combined 1:4 with 50% methanol to obtain a final dilution of D20. |
Combined analysis:
| Analysis ID | AN003741 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Thermo Syncronis HILIC (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | AUC |
Chromatography:
| Chromatography ID: | CH002772 |
| Methods Filename: | culture_protocol_kb.docx |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Syncronis HILIC (100 x 2.1mm,1.7um) |
| Column Temperature: | 30C |
| Flow Gradient: | 2 stage linear gradient |
| Flow Rate: | 600uL/min |
| Sample Injection: | 2uL |
| Solvent A: | 100% water; 20 mM ammonium formate, pH 3 |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Analytical Time: | 15 minutes |
| Washing Buffer: | 10% MeOH in H2O + 0.1% formic acid |
| Target Sample Temperature: | 4 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003489 |
| Analysis ID: | AN003741 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | 50-750 m/z, peak integration and assignment using El-Maven software matching to authentic standard library (~150 compounds). |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | culture_protocol_kb.docx |
