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MB Sample ID: SA001679

Local Sample ID:LNP126
Subject ID:SU000050
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:NOD/ShiLtj
Animal Animal Supplier:Jackson Laboratories
Animal Housing:SPF (Helicobacter neg / MNV neg)
Animal Feed:normal chow
Species Group:Mammal

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Sample Preparation:

Sampleprep ID:SP000046
Sampleprep Summary:Frozen liver samples were weighed (50-100 mg) into labeled homogenizer bead tubes. Cold acetonitrile:water (50%) was added to tissue based on weight to make 1 mg/mL homogenates. The samples were extracted and homogenized on a Spex Geno/Grinder for two 45 second pulses at 1750 rpm. Samples were centrifuged at 12000 rcf for 5 min. Liver supernatants were transferred into BSI-labeled tubes. A 500 μL aliquot/sample was transferred into a second set of BSI-labeled tubes for further processing. To make pooled samples, a 300 μL aliquot of selected TranSTAT liver supernatants was combined in a 2 mL tube and used for QC samples in DuraSTAT, TranSTAT, EstroSTAT and NOD sub-studies. Additionally, a 200 μL aliquot of selected VG STAT liver supernatants was combined in a separate 2 mL tube and used as QC samples in the VG STAT sub-study. Three 500 μL aliquots were transferred into BSI-labeled tubes for each set of Pooled QC samples. All samples were then dried on an Eppendorf rotaVap (V-AL setting) at 30°C until dry and stored at -80 °C. On the second day of processing, 630 μL of D2O was added into each dried liver extract tube. Chenomx Internal Standard solution (Chenomx ISTD, Edmonton, Alberta, Canada) contains 5mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS, Chemical Shift Indicator), 100 mM Imidazole (pH indicator), and 0.2% NaN3 (to inhibit bacterial growth) was added (70 µl), and the samples were vortexed on a multi-tube vortexer for 10 minutes at speed 5. Tubes were then centrifuged at 12000 rcf for 5 minutes and a 600 μL aliquot of the supernatant was transferred into 5mm NMR tubes (Wilmad LabGlass, New Jersey, USA) which were kept on ice until data acquisition.
Sampleprep Protocol Filename:NOD_Liver_Metabolomics_Procedure.docx