Return to study ST000083 main page
MB Sample ID: SA004272
| Local Sample ID: | SBEP_Microbiome.022 |
| Subject ID: | SU000102 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | 129/SvJ |
| Age Or Age Range: | 6- to 8-week-old |
| Gender: | Female |
| Animal Animal Supplier: | Jackson Laboratories,Bar Harbor, ME |
| Animal Housing: | specific pathogen-free conditions in filter-top cages |
| Animal Feed: | food provided ad libitum |
| Animal Water: | sterile water |
| Species Group: | Mammals |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000102 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | 129/SvJ |
| Age Or Age Range: | 6- to 8-week-old |
| Gender: | Female |
| Animal Animal Supplier: | Jackson Laboratories,Bar Harbor, ME |
| Animal Housing: | specific pathogen-free conditions in filter-top cages |
| Animal Feed: | food provided ad libitum |
| Animal Water: | sterile water |
| Species Group: | Mammals |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SBEP_Microbiome.022 | SA004272 | FL001212 | Infected | Infection |
| SBEP_Microbiome.022 | SA004272 | FL001212 | Experimental 1 | Experimental Group |
| SBEP_Microbiome.022 | SA004272 | FL001212 | 14 | Harvest Day |
Collection:
| Collection ID: | CO000085 |
| Collection Summary: | Feces collected and frozen at seven time points post infection and one time point pre-infection |
| Collection Protocol Comments: | On the day prior to infection (day -1), and at seven time points post-infection, mice from one cage were transferred to a clean cage and fecal samples produced at that time were collected, pooled, and immediately frozen at -80° C. Samples were collected on days -1, 1, 3, 6, 10, 14, 21, and 28. |
| Sample Type: | Feces |
| Collection Method: | Fecal matter collection |
| Collection Location: | mice from one cage were transferred to a clean cage and fecal samples produced |
| Collection Frequency: | days -1, 1, 3, 6, 10, 14, 21, and 28 relative to day of infection (day 0) |
| Collection Time: | days -1, 1, 3, 6, 10, 14, 21, and 28 relative to day of infection (day 0) |
| Volumeoramount Collected: | four to 25 fecal pellets per pooled sample |
| Storage Conditions: | frozen at -80° C |
Treatment:
| Treatment ID: | TR000103 |
| Treatment Summary: | Experimental: Infected with 1.6 x 10^8 CFU S. Typhimurium | mouse | Control: treated with equal volume saline solution |
| Treatment Protocol Comments: | A final inoculum of 1.6 x 10^8 CFU S. Typhimurium/mouse was delivered by oral gavage to 10 mice (two cages of five mice each = Salmonella-infected). An equal number of mock-infected animals (two cages of five mice each = control) were administered an equal volume of sterile saline. Our infecting dose (10^8 CFU/mouse) aimed to establish a persistent infection that would ensure observation of S. Typhimurium proteins in downstream analyses. |
| Treatment: | Biotic / Abiotic |
| Treatment Compound: | S. Typhimurium / Saline |
| Treatment Dose: | 1.6 x 10^8 CFU S. Typhimurium/mouse / equal volume saline solution |
| Treatment Vehicle: | Saline |
| Animal Fasting: | 14 h before orogastric inoculation |
| Animal Endp Euthanasia: | Carbon Dioxide Asphixiation followed by Cervical Dislocaton |
| Animal Endp Tissue Coll List: | Feces |
| Animal Endp Tissue Proc Method: | Homogenization |
Sample Preparation:
| Sampleprep ID: | SP000098 |
| Sampleprep Summary: | Feces thawed, buffer added, vortexed, filtered and centrifuged after which supernatant subjected to further centrifugation and chemical derivatization |
| Sampleprep Protocol Comments: | After thawing, 150 mM ammonium bicarbonate buffer was added to the sample (between 1-2.5 ml based upon starting weight; volumes were recorded and used for downstream normalization), which was subsequently vortexed to disrupt fecal pellets. The resulting slurry was filtered through a 70 mm sieve to separate and remove large debris (mostly undigested food particles). Filtrate was centrifuged (900 x g for 10 min), and the protein-rich pellet thought to contain cellular material was retained as P1. The supernatant was centrifuged to further clarify the sample (15,000 x g for 10 min). The pellet was retained as P2 and the supernatant retained as SN2. All chemicals and reagents used in metabolomics analyses were purchased from Sigma-Aldrich (St. Louis, MO), except for ammonium bicarbonate (Merck, Darmstadt, Germany), mixture of fatty acid methyl esters (FAMEs) and deuterated myristic acid (Agilent Technologies, Santa Clara, CA). Deionized and purified water was used to prepare buffer and standard solutions (Nanopure Infinity ultrapure water system, Barnstead, Newton, WA). SN2 samples (see Fecal sample preparation) were transferred to 0.6 ml microcentrifuge tubes, and water soluble metabolites were extracted with four volumes of chilled (-20° C) chloroform: methanol mixture (2:1). After separating the two phases via centrifugation (12,000 x g, 5 min), the upper aqueous layers were transferred to glass vials and dried under a vacuum concentrator (SpeedVac; Thermo Scientific, Waltham, MA). All extracted metabolites were subjected to chemical derivatization to enhance their stability and volatility during GC-MS analysis. Methoxyamine in pyridine (30 mg/ml) was added to each dried sample, and incubated at 37° C with shaking for 90 min to protect carbonyl groups and reduce the number of tautomeric peaks. N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) was then added, followed by incubation at 37° C with shaking for 30 min to transform hydroxyl and amine groups to trimethylsilyated (TMS) forms. The samples were then allowed to cool to room temperature and were analyzed using gas chromatography (GC)-MS. |
| Processing Method: | Homogenization, Filtration, Centrifugation |
| Extraction Method: | SN2 samples (see Fecal sample preparation) were transferred to 0.6 ml microcentrifuge tubes, and water soluble metabolites were extracted with four volumes of chilled (-20° C) chloroform: methanol mixture (2:1). After separating the two phases via centrifugation (12,000 x g, 5 min), the upper aqueous layers were transferred to glass vials and dried under a vacuum concentrator (SpeedVac; Thermo Scientific, Waltham, MA). |
| Extract Concentration Dilution: | chloroform: methanol mixture (2:1) |
| Extract Enrichment: | dried under a vacuum concentrator |
| Extract Storage: | dried under a vacuum concentrator |
| Sample Resuspension: | Methoxyamine in pyridine (30 mg/ml) |
| Sample Derivatization: | Methoxyamine in pyridine (30 mg/ml), N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
Combined analysis:
| Analysis ID | AN000135 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890A |
| Column | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5975C |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH000095 |
| Chromatography Summary: | Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation |
| Chromatography Comments: | Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min. |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| Flow Rate: | 0.450.5 mL/min |
| Injection Temperature: | 250 C |
| Sample Injection: | 1 L, splitless |
| Analytical Time: | 37.5 min |
| Oven Temperature: | 60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C |
| Sample Syringe Size: | 10 L |
| Chromatography Type: | GC |
MS:
| MS ID: | MS000111 |
| Analysis ID: | AN000135 |
| Instrument Name: | Agilent 5975C |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis. |
| Ion Mode: | POSITIVE |
| Scan Range Moverz: | 50-550 m/z |
