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MB Sample ID: SA051028
| Local Sample ID: | 141215bctsa25_1 |
| Subject ID: | SU000912 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Fifth-generation C57/BL6 Pkd1tm2Ggg (Piontek et al., 2004) mice were crossed to the reporter mice C57/BL6 congenic B6.129S4-Gt(ROSA)26Sortm1Sor/J (stock 003474, Jackson Laboratories) and to C57/BL6 tamoxifen-Cre (B6.Cg-Tg(Cre/Esr1)5Amc/J mice (stock 004682), Jackson Laboratories) or Ksp-Cre B6.Cg-Tg(Cdh16-cre)91Igr/J(stock 012237), Jackson Laboratories). |
| Gender: | Males |
| Animal Animal Supplier: | Jackson Laboratories |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000912 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Fifth-generation C57/BL6 Pkd1tm2Ggg (Piontek et al., 2004) mice were crossed to the reporter mice C57/BL6 congenic B6.129S4-Gt(ROSA)26Sortm1Sor/J (stock 003474, Jackson Laboratories) and to C57/BL6 tamoxifen-Cre (B6.Cg-Tg(Cre/Esr1)5Amc/J mice (stock 004682), Jackson Laboratories) or Ksp-Cre B6.Cg-Tg(Cdh16-cre)91Igr/J(stock 012237), Jackson Laboratories). |
| Gender: | Males |
| Animal Animal Supplier: | Jackson Laboratories |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 141215bctsa25_1 | SA051028 | FL009824 | control | Treatment* |
Collection:
| Collection ID: | CO000906 |
| Collection Summary: | Kidneys from two Pkd1cko/cko animals (121112-C: male, bP12; and 94414: male, P463) were harvested, minced and digested using a collagenase/hyaluronidase solution (Stemcell technologies, cat. no. 07912) followed by proximal or collecting/distal tubule cell enrichment using, respectively, biotinylated Lotus tetragonolobus Lectin (LTL) (Vector Laboratories, cat. no. B-1325) or biotinylated Dolichos biflorus Agglutinin (DBA) (Vector Laboratories, ca. no. B-1035) and Cellection Biotin Binder kit (ThermoFischer, cat. no. 11533D). Cells were immortalized using the large T antigen (Addgene plasmid no. 22298). Pkd1 was conditionally inactivated using cre recombinase (121112C-LTL cells; Excellgen, cat. no. EG-1001) or viral transduction (121112-C DBA and 94414-LTL/DBA cells) using LV-Cre (Addgene plasmid no. 12106). At the time of inactivation, a corresponding control was generated using viral transductionwith plasmid LV-Lac (Addgene plasmid no. 12108). Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1). mCCDcl1 (mCCD) cells were a kind gift from the Rossier lab (Gaeggeler et al., 2005). mCCD Pkd1 knock-down cells were generated using viral transduction with the shRNA clone TRCN0000072085 and the corresponding pLKO.1 TRC21 control (Addgene plasmid 10,879). Cells were grown in DMEM/F12 media (Life cat. no. 21041–025) with 2% FBS (GEMINI Bio-Products cat. no. 100–106), 1X Insulin-Transferrin-Selenium (Thermo Fisher Scientific, cat. no. 41400–045), 5 uM dexamethasone (SIGMA, cat. no. D1756), 10 ng/ml EGF (SIGMA, cat. no. SRP3196), 1 nM 3,3′,5-Triiodo-L-thyronine (SIGMA, cat. no. T6397) and 10 mM HEPES (CORNING, cat. no. 25-060-CI). Mouse embryonic fibroblasts (MEFs) were obtained from E12.5 and E13.5 Pkd1 knockout (Pkd1tm1GGG (Bhunia et al., 2002)) and control mice. Briefly, whole embryos were minced, washed in PBS and cultured in six-well tissue culture plate in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). A total of 6 MEF lines was used for this study: three from E13.5 mouse littermates (2 Pkd1ko/ko and 1 Pkd1wt/wt) immortalized using large T antigen (Addgene plasmid no. 22,298) and an additional set of three primary (passage 2, non-immortalized) E12.5 embryos (1 Pkd1ko/ko and 2 Pkd1wt/wt littermate controls). |
| Sample Type: | Tissue |
Treatment:
| Treatment ID: | TR000926 |
| Treatment Summary: | Metabolomics analysis of 14 male kidneys, 8 ADPKD mutants and 6 controls. |
Sample Preparation:
| Sampleprep ID: | SP000919 |
| Sampleprep Summary: | 1. Weigh 50 mg tissue sample in to a 25 ml conical polypropylene centrifuge tube. 2. Add 2.5mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent. 3. Centrifuge the samples at 2500 rpm. for 5 minutes. Aliquot 2 X 500μl supernatant, one for analysis and one for a backup sample. Store backup aliquot in the -20°C freezer. 4. Evaporate one 500μl aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness 5. The dried aliquot is then re-suspended with 500l 50% acetonitrile (degassed as given) 6. Centrifuge for 2 min at 14000 rcf using the centrifuge Eppendorf 5415. 7. Remove supernatant to a new Eppendorff tube. 8. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. 9. Submit to derivatization. |
| Sampleprep Protocol Filename: | SP_SOP_Extraction_of_Mammalian_Tissue.pdf |
Combined analysis:
| Analysis ID | AN001428 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 6890N |
| Column | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
| MS Type | EI |
| MS instrument type | GC-TOF |
| MS instrument name | Leco Pegasus III GC TOF |
| Ion Mode | POSITIVE |
| Units | Counts |
Chromatography:
| Chromatography ID: | CH000999 |
| Instrument Name: | Agilent 6890N |
| Column Name: | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
| Column Pressure: | 7.7 PSI (initial condition) |
| Column Temperature: | 50 - 330°C |
| Flow Rate: | 1 ml/min |
| Injection Temperature: | 50°C ramped to 250°C by 12°C/s |
| Sample Injection: | 0.5 uL |
| Oven Temperature: | 50°C for 1 min, then ramped at 20°C/min to 330°C, held constant for 5 min |
| Transferline Temperature: | 230°C |
| Washing Buffer: | Ethyl Acetate |
| Sample Loop Size: | 30 m length x 0.25 mm internal diameter |
| Randomization Order: | Excel generated |
| Chromatography Type: | GC |
MS:
| MS ID: | MS001318 |
| Analysis ID: | AN001428 |
| Instrument Name: | Leco Pegasus III GC TOF |
| Instrument Type: | GC-TOF |
| MS Type: | EI |
| Ion Mode: | POSITIVE |
| Ion Source Temperature: | 250°C |
| Ionization Energy: | 70eV |
| Mass Accuracy: | Nominal |
| Scan Range Moverz: | 85-500 |
| Scanning Cycle: | 17 Hz |
| Scanning Range: | 80-500 Da |
| Skimmer Voltage: | 1850 |
