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MB Sample ID: SA001678

Local Sample ID:LNC140
Subject ID:SU000050
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:NOD/ShiLtj
Gender:Male
Animal Animal Supplier:Jackson Laboratories
Animal Housing:SPF (Helicobacter neg / MNV neg)
Animal Feed:normal chow
Species Group:Mammal

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Subject:

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

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
LNC140SA001678FL000566ControlTreatment Group

Collection:

Collection ID:CO000033
Collection Summary:-
Sample Type:liver
Collection Time:6 weeks

Treatment:

Treatment ID:TR000051
Treatment Summary:Three mice were exposed to PAT and 3 mice were non-exposed controls. PAT= therapeutic dose-pulsed antibiotic treatment.
Treatment Compound:PAT

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

Analysis:

MB Sample ID:SA001678
Analysis ID:AN000053
Laboratory Name:RTI/ DHHMRI
Analysis Type:NMR
Analysis Comments:NMR (700 MHz)
Acquisition Date:41527
Software Version:Top Spin 3.2
Operator Name:Wimal Pathmasiri/ Kevin Knagge/ Jason Winnikie
Randomization Order:yes
Detector Type:NMR
Data Format:NMR
Chromatography ID:CH000034
Num Factors:3

NMR:

NMR ID:NM000015
Analysis ID:AN000053
Instrument Name:Bruker Avance III
Instrument Type:FT-NMR
NMR Experiment Type:1D 1H
Field Frequency Lock:Deuterium
Standard Concentration:0.5mM
Spectrometer Frequency:700 MHz
NMR Probe:cyrogenically cooled ATMA inverse probe
NMR Solvent:D2O
NMR Tube Size:5mm x 4inch
Shimming Method:topshim
Pulse Sequence:noesypr1d
Water Suppression:presat
Pulse Width:14.84 us
Power Level:25.704w
Receiver Gain:10
Offset Frequency:3293 Hz
Chemical Shift Ref Cpd:DSS
Temperature:298.1 K
Number Of Scans:64
Dummy Scans:4
Acquisition Time:2.3243434
Relaxation Delay:2 s
Spectral Width:20.1358
Num Data Points Acquired:65536
Real Data Points:65536
Line Broadening:0.5
Zero Filling:yes
Apodization:lorentzian
Baseline Correction Method:polynomial
Chemical Shift Ref Std:DSS
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