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MB Sample ID: SA004265
| Local Sample ID: | SBEP_Microbiome.015 |
| 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: | Mammal |
Select appropriate tab below to view additional metadata details:
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) |
