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| Status |
Public on Dec 04, 2009 |
| Title |
70 |
| Sample type |
genomic |
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| Source name |
lymphoblast from Coriell family 1990
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| Organism |
Homo sapiens |
| Characteristics |
cell type: lymphoblast from Coriell family 1990
cell amplication: single cell amplified by MDA
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cells were isolated from buccal swabs, semen samples, adult blood, immortalized cell lines, and Day 3 cryopreserved embryos. Single tissue culture (lymphocytes) and buccal cells were isolated using a sterile stripper tip (Midatlantic Diagnostic, Mt. Laurel, NJ, USA) affixed to a pipette (Drummond Scientific, Broomall, PA, USA) and a stereoscope (Leica, Wetzlar, Germany). Embryos were thawed, and then individual blastomeres were separated using a micromanipulator (Transferman NK2-Eppendorf, Westbury, NY, USA) after zona pellucida drilling using acidified Tyrode’s solution. Sperm cells were manually isolated using a micromanipulator (Transferman NK2-Eppendorf). Aside from sperm, single cells were washed sequentially four times with wash buffer (5.6mg/ml KCl, 6mg/ml bovine serum albumin). Two different lysis/amplification protocols were used in the analysis: (i) Rubicon Whole Genome Amplification (Ann Arbor, MI, USA), and (ii) multiple displacement amplification (MDA) with Proteinase K Buffer (PKB). Protocol (i) was performed according to the manufacturer’s instructions. For protocol (ii), cells were placed in 5µl PKB (Arcturus PicoPure Lysis Buffer, 50mM DTT), incubated at 56°C for one hour, and then heat inactivated at 95°C for ten minutes. MDA reactions were incubated at 30°C for 2.5 hours and then 65°C for five minutes. Genomic DNA from bulk tissue (Epicentre MasterAmp Buccal Swabs, Madison, WI, USA) was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). No template controls (buffer blanks) were performed for each amplification method. All buffer blanks produced intensities equivalent to the noise floor (an intensity of 1000 in the 95th percentile on the green detection channel).
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| Label |
C-Bio and A-DNP
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| Label protocol |
as per Illumina's instructions except reduced amplification time to 6 hours
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| Hybridization protocol |
as per Illumina's instructions except reduced time to 6 hours
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| Scan protocol |
as per Illumina's instructions
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| Description |
4554318307_R04C01
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| Data processing |
Genotypes are inherently inaccurate in single cell microarray data due to biases introduced during multiple displacement amplification. Therefore genotypes were not produced, and instead raw intensity data was used to build probabilistic models under the framework of the Parental Support algorithm.
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| Submission date |
Dec 01, 2009 |
| Last update date |
Dec 03, 2009 |
| Contact name |
David Scott Johnson |
| E-mail(s) |
djohnson@genesecurity.net
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| Phone |
415-978-2101
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| Organization name |
Gene Security Network
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| Street address |
2686 Middlefield Road, Suite C
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| City |
Redwood City |
| State/province |
CA |
| ZIP/Postal code |
94063 |
| Country |
USA |
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| Platform ID |
GPL8855 |
| Series (1) |
| GSE19247 |
Preclinical Validation of a Microarray Method for Full Molecular Karyotyping of Blastomeres in a 24-hour Protocol |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM477142_4554318307_R04C01_1.xml.gz |
618 b |
(ftp)(http) |
XML |
| GSM477142_4554318307_R04C01_2.xml.gz |
611 b |
(ftp)(http) |
XML |
| GSM477142_4554318307_R04C01_3.xml.gz |
612 b |
(ftp)(http) |
XML |
| GSM477142_4554318307_R04C01_4.xml.gz |
609 b |
(ftp)(http) |
XML |
| GSM477142_4554318307_R04C01_Grn.idat.gz |
1.9 Mb |
(ftp)(http) |
IDAT |
| GSM477142_4554318307_R04C01_Red.idat.gz |
1.9 Mb |
(ftp)(http) |
IDAT |
| Processed data not applicable for this record |
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