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| Status |
Public on Mar 01, 2015 |
| Title |
MI-AML137T |
| Sample type |
genomic |
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| Source name |
primary human: blood or bone marrow, FACS sort-purified AML blasts
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| Organism |
Homo sapiens |
| Characteristics |
disease state: non-FAB-M3 AML
individual: MIAML137
paired sample: tumor
cell type: FACS sort-purified AML blasts
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| Treatment protocol |
n/a
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
[purification protocol] AML blast DNA used for SNP 6.0 profiling was extracted from negatively column enriched AML cell samples as described that were further purified as follows: post-Miltenyi column samples were washed and stained with FITC-conjugated anti-CD33, PE-conjugated anti-CD13, and APC-conjugated anti-CD45 (all antibodies: eBioscience, San Diego, CA). After final washing, propidium iodide (PI) was added to a concentration of 1 μg/ml to discriminate dead cells. Sorting of cells was done on a FACS-ARIA high-speed flow cytometer (Becton Dickinson, Mountain View, CA). Live cells (PI-negative) were gated for blasts by identifying those cells with intermediate-intensity staining for CD45 and low- to moderate-intensity side scatter. CD33 and CD13 were then used in order to further discriminate myeloid blasts versus contaminating erythroid lineage or other non-myeloid cells. Cells forming a dense population cloud on the CD33 versus CD13 plots that were either single marker positive or double positive were sorted. Aliquots of final cell preparations were made for the majority of sorted samples and manually analyzed for cell composition using cytospins and blast counts.
Highly pure blast cells or buccal swabs were digested overnight in 100 mM Tris pH 8.0, 50 mM EDTA, 50 mM NaCl, 0.5% SDS and 100 μg/ml of Proteinase K (Invitrogen) at 560C. DNA was extracted using phenol-chloroform and precipitated using ammonium acetate, ethanol and glycogen.
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| Label |
biotin
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| Label protocol |
The DNA was prepared for hybridization to Affymetrix SNP 6.0 arrays according to the manufacturer's recommendations.
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| Hybridization protocol |
Affymetrix Hybridization Oven 640 for 16 hrs at 50 deg C with rotation at 60 rpm. Washing and staining was done on an Affymetrix Fluidics Station 450.
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| Scan protocol |
Scanning was done using an Affymetrix Scanner 3000 7G with autoloader.
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| Data processing |
The DNA was prepared for hybridization to SNP 6.0 arrays according to the manufacturer’s recommendations. Affymetrix CEL files for each blast and buccal sample were analyzed using Genotyping Console software for initial quality control, followed by use of the Affymetrix “Birdseed” algorithm to generate tab-delimited SNP call files in text format. Call rates for the entire group of samples included in this report were between 93.64% and 99.45%, with a mean call rate of 98.17%; none of the tumor DNA samples gave out-of-bounds results.
Sample copy number heatmap displays were obtained from CEL files through use of the freely available software dChip, adapted to run on a 64-bit computer environment. To generate functional and practical displays of LOH, a two-step, internally developed, Java-based software analysis system was employed. The Pre-LOH Unification Tool (PLUT) served to align all individual patient SNP calls to their respective dbSNP rs ID numbers and genomic physical positions prior to incorporation into the LOH tool version 2, an updated version of the LOH tool able to accommodate Affy SNP 6.0 array data
[primary data description] dChip SNP software (www.dchip.org), build date Feb 25, 2009, compatible with 64-bit OS, analyzed the CEL files. CEL files were normalized using the invariant set method, followed by model-based expression index calculations, and finally inferred copy number data were generated using a median-smoothing algorithm. See supplementary file linked to Series record.
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| Submission date |
Sep 11, 2014 |
| Last update date |
Mar 01, 2015 |
| Contact name |
Sami N Malek |
| E-mail(s) |
smalek@med.umich.edu, pouillet@med.umich.edu
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| Phone |
734-763-1222
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| Organization name |
University of Michigan
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| Department |
Internal Medicine, Hematology-Oncology
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| Street address |
4410 Cancer Center; 1500 E Medical Center Dr
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL6801 |
| Series (1) |
| GSE61323 |
Integrated Genomic Profiling, Therapy Response And Survival In Adult Acute Myelogenous Leukemia |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1502237_Malek_MI-AML137-T.CEL.gz |
33.2 Mb |
(ftp)(http) |
CEL |
| GSM1502237_Malek_MIAML137-T.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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