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| Status |
Public on Dec 04, 2013 |
| Title |
line D fibroblast 22_21 |
| Sample type |
genomic |
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| Source name |
fibroblast
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| Organism |
Homo sapiens |
| Characteristics |
cell type: fibroblast
gender: female
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| Treatment protocol |
none
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| Growth protocol |
cells were grown according to standard conditions (Brennand, et al. Nature 2011)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cells were transferred to a 200uL PCR tube containing 1.5 uL lysis buffer alkaline lysis buffer. Mulieple Displacement amplification was perfomred according to manufacturers instructions (Genomiphi V2, GE Helthsciences).MDA products (5 ng) were examined for even amplification (e.g., +/- 5% of the Ct for 5 ng bulk genomic DNA) using qPCR (Applied Biosystems, San Diego, CA). To test for even amplification, we used a 10 locus subset of the 47 single copy loci used in Hosono et al. (Genome Research, 2009) (Chr1p, Chr2p, Chr3q, Chr7p, Chr10p, Chr11p, Chr14q, Chr17q, Chr19p, and Chr21q).
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| Label |
biotin
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| Label protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
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| Hybridization protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
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| Scan protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
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| Description |
single cell genome was subjected to multiple displacement amplification prior to hybridization
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| Data processing |
Partek Genomics Suite Software (version 6.6 beta, Partek, St. Louis, MO) was used to calculate predicted copy numbers for each probe set intensity. A custom copy number model composed of 161 MDA single cell experiments (from this and other studies) was generated to perform quantile normalization of the calculated copy numbers. The background-adjusted values were then subjected to GC correction in windows of 10 Mb, and artifact-prone probes were removed according to Pugh et al. (Nuleic Acids Research, 2008) We then performed smoothing by taking the median copy number value in non-overlapping genomic windows composed of 100 probes. On average, each 100-probe bin corresponds to 666 Kb of genome sequence. At this stage we also excluded 6 of 107 samples that had excessively "noisy" copy number profiles, defined as having a median absolute deviation (MAD) greater than 0.7. To detect CNVs, we used the circularly binary segmentation (CBS) algorithm from the DNAcopy package in R, with the following parameters: alpha=0.001, undo.splits="sdundo", undo.SD=1. We defined CNVs as segments composed of 10 or more contiguous genomic windows whose copy number value differed from the dataset's median copy number by at least 1 MAD. We did not attempt to detect CNVs on the Y chromosome.
Single cell genomes, amplified by mulitple displacement amplification, were analyzed on Affy 250K_NSP arrays.
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| Submission date |
Oct 22, 2013 |
| Last update date |
Dec 04, 2013 |
| Contact name |
Michael J McConnell |
| E-mail(s) |
mikemc@virginia.edu
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| Organization name |
University of Virginia
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| Street address |
1340 Jefferson Park Ave
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| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
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| Platform ID |
GPL3718 |
| Series (1) |
| GSE51538 |
SNP array analysis of single cell genomes |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1247554_11640.McConnell.DF-22.sh_Mapping250K_Nsp_.CEL.gz |
23.6 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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