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MB Sample ID: SA210139

Local Sample ID:Terence_MS10_Quad_Aq
Subject ID:SU002275
Subject Type:Mammal
Subject Species:Mus musculus
Genotype Strain:C57BL/6J
Age Or Age Range:13 weeks
Weight Or Weight Range:(28.4±1.1 g (control SHAM mice) vs 26.4±2.1 g (control AVF mice) vs 23.6±2.1 (CKD SHAM mice) vs 24.7±1.3 (CKD AVF mice), N=8-10/group).
Gender:Male
Animal Animal Supplier:Jackson Labs (Stock # 000664)
Animal Housing:Housed in a temperature of 22 oC
Animal Light Cycle:12-hour light/12-hour dark
Animal Feed:Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD)
Animal Water:free access to food and water (3-5 animals per cage).
Animal Inclusion Criteria:(3-5 animals per cage).

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Subject:

Subject ID:SU002275
Subject Type:Mammal
Subject Species:Mus musculus
Genotype Strain:C57BL/6J
Age Or Age Range:13 weeks
Weight Or Weight Range:(28.4±1.1 g (control SHAM mice) vs 26.4±2.1 g (control AVF mice) vs 23.6±2.1 (CKD SHAM mice) vs 24.7±1.3 (CKD AVF mice), N=8-10/group).
Gender:Male
Animal Animal Supplier:Jackson Labs (Stock # 000664)
Animal Housing:Housed in a temperature of 22 oC
Animal Light Cycle:12-hour light/12-hour dark
Animal Feed:Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD)
Animal Water:free access to food and water (3-5 animals per cage).
Animal Inclusion Criteria:(3-5 animals per cage).

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Terence_MS10_Quad_AqSA210139FL025644CKD_SHAMFactor1***

Collection:

Collection ID:CO002268
Collection Summary:Two weeks after surgery, mice were anesthetized using isoflurane and the left quadriceps muscle was rapidly dissected and immediately frozen in liquid nitrogen and stored at -80oC for metabolite extraction. Euthanasia was carried out by thoracotomy followed by cervical dislocation.
Sample Type:Muscle
Collection Method:While under isoflurane anesthesia, tissues were rapidly dissected and snap frozen in liquid nitrogen and stored at -80°C until metabolite extraction
Collection Location:University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, Gainesville, FL 32611
Storage Conditions:-80℃
Storage Vials:cryovials

Treatment:

Treatment ID:TR002287
Treatment Summary:Briefly, mice were acclimated to a standard casein-based chow (Control Diet) for 7 days. Thereafter, the animals were randomly assign to either remain on Control Diet (non-CKD groups) or receive adenine-supplemented diet to induce CKD. Following three weeks of the casein/adenine diet, animals were randomly assigned to receive either an arteriovenous fistula surgery (AVF) or sham surgery (SHAM)
Animal Vet Treatments:none
Animal Anesthesia:isoflurane
Animal Fasting:non-fasted
Animal Endp Euthanasia:Euthanasia was carried out by thoracotomy followed by cervical dislocation.
Animal Endp Tissue Coll List:Skeletal muscle (quadriceps)

Sample Preparation:

Sampleprep ID:SP002281
Sampleprep Summary:Weights of frozen quadriceps specimens were weighed using a microbalance (Mettler-Toledo, Columbus, OH, USA). Next, a slightly modified FOLCH extraction was performed to extract aqueous and lipid phase metabolites. The aqueous phase was lyophilized overnight (Labconco Corporation, Kansas, MO, USA) and the lipid phase was dried by passing inert nitrogen gas. The resulting aqueous and lipid phase dry powders were stored at -80oC until analysis using nuclear magnetic resonance (NMR). The dry powder of aqueous phase samples was dissolved in 50 µL of phosphate buffer system (50 mM, pH 7.2) consisting of 0.5 mM D6-DSS, 2 mM EDTA and 0.2% NaN2. Lipid phase dry powders were dissolved in 70 µL of CDCl3 supplemented with 10 mM of pyrazine (as internal NMR standard). All samples were loaded into 1.5 mm optical density (O.D.) NMR tubes.
Sampleprep Protocol Filename:AVF_NMR_Aqueous_Phase_Procedures.docx
Processing Method:Lyophilization and Homogenization
Processing Storage Conditions:-80℃
Extraction Method:Modified FOLCH extraction
Extract Storage:-80℃
Sample Resuspension:In 50 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS and 0.2% sodium azide for aqueous phase samples.
Sample Spiking:0.5 mM of DSS for aqueous phase samples

Analysis:

MB Sample ID:SA210139
Analysis ID:AN003583
Laboratory Name:Terence lab, UF
Analysis Type:NMR
Acquisition Date:09/14/2021
Software Version:Bruker Topspin
Operator Name:Ram Khattri
Detector Type:Bruker
Data Format:fid, 1r
Num Factors:5
Num Metabolites:37
Units:mM

NMR:

NMR ID:NM000241
Analysis ID:AN003583
Instrument Name:Bruker Avance III Cryo800 MHz 54 mm
Instrument Type:FT-NMR
NMR Experiment Type:1D-1H
Field Frequency Lock:Deuterium
Standard Concentration:0.5 mM DSS
Spectrometer Frequency:799.9
NMR Probe:CP TXI CryoProbe
NMR Solvent:Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in deuterated environment
NMR Tube Size:1.5 mm O.D.
Shimming Method:Topshim
Pulse Sequence:noesypr1d
Water Suppression:presat
Pulse Width:90-degree
Receiver Gain:645
Offset Frequency:2827.31 Hz
Chemical Shift Ref Cpd:DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid)
Temperature:25 o C
Number Of Scans:128 scans
Dummy Scans:8
Acquisition Time:4 s
Relaxation Delay:1 s
Spectral Width:9523.8
Num Data Points Acquired:38094
Real Data Points:65536
Line Broadening:0.22 Hz
Zero Filling:65,536 points
Apodization:Exponential
Baseline Correction Method:Spline
Chemical Shift Ref Std:0 ppm for DSS
Binned Increment:None
Binned Data Excluded Range:None
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