|
| Status |
Public on Jun 06, 2007 |
| Title |
GM12872 |
| Sample type |
RNA |
| |
|
| Source name |
lymphoblastoid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
Coriell cell line repository identifier: GM12872
http://locus.umdnj.edu/nigms/nigms_cgi/display.cgi?GM12872
CEPH sample
Gender: Male
Associated family: 1459-9
Family relationship: paternal grandfather
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM12872
|
| Treatment protocol |
Untreated, baseline expression data.
|
| Growth protocol |
Each sample was collected while in exponential growth.
Lymphoblastoid cell lines were centrifuged at 400 × g to remove media and 5 mL fresh lymphoblastoid cell media (LCL media) containing RPMI 1640 (Mediatech)/1% l-glutamine (Mediatech) plus 20% FBS (HyClone Laboratories) for the initial passage and then passaged every 48 h with LCL medium and 15% FBS. Cell suspensions were transferred to 25 cm2 flasks and incubated at 37oC in a 90% humidified 5% CO2 atmosphere. Cell lines were maintained at a concentration of 3.5-4.0×105 cells/mL and harvested following the 4th passage, only if viability >= 85%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lymphoblastoid cell line suspensions were spun at 400 × g for 5 min to remove media. Cell pellets were washed twice with ice-cold PBS and stored at -80°C. Total RNA was extracted using RNeasy Plus Kits according to the manufacturers protocol.
|
| Label |
biotin
|
| Label protocol |
Ribosomal RNA was depleted from 1µg of total RNA using the RiboMinus Human/Mouse Transcriptome Isolation kit (Invitrogen Corp., Carlsbad, CA). cDNA was generated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Inc., Santa Clara, CA) per manufacturer's instructions. cDNA was fragmented and end labeled using the GeneChip® WT Terminal Labeling Kit (Affymetrix, Inc.).
|
| |
|
| Hybridization protocol |
Approximately 5.5ug of labeled DNA target was hybridized to the Affymetrix GeneChip Human Exon 1.0 ST Array at 45oC for 16 hours per manufacturers recommendation (see http://www.affymetrix.com/products/arrays/exon_application.affx for additional information). Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450.
|
| Scan protocol |
All samples were scanned on a GCS3000 Scanner (Affymetrix, Inc.).
|
| Description |
Gene expression on 176 HapMap cell lines (87 CEU and 89 YRI) was determined using the Affymetrix GeneChip Human Exon 1.0 ST Array.
|
| Data processing |
dbSNP in Probe Filtering and Background Correction Protocol: The start and end coordinates of all probes of the Exon array were queried against the human genome (hg17). The coordinates for all SNPs were then queried in the dbSNP database (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/projects/SNP/) (release 126) and then based on location, we identified probes harboring SNPs. In total, ~400,000 probes contained SNPs within their structure. Of the ~1.4 million probesets of the exon array, 255,676 contained at least one probe with SNP. Among these affected probesets, 105,000 harbored 2 or more probes with SNPs. Resulting probe signal intensity files were filtered by removing this group of 105,000probesets. After filtering, individual probe intensities were background corrected by subtracting the median intensity of a population of non-genomic probes with the same GC content to account for any non-specific hybridization. The resulting probe signal intensities were sketch quantile normalized over all 176 samples and gene-level expressions were summarized using the robust multi-array average (RMA) method with signals generated on a core set of exons. A constant of 16 was added to all probeset intensities for variance stabilization and summarized signals were then log2 transformed with a median polish.
|
| |
|
| Submission date |
May 14, 2007 |
| Last update date |
Apr 26, 2009 |
| Contact name |
Eileen Dolan |
| E-mail(s) |
edolan@medicine.bsd.uchicago.edu
|
| Phone |
773-702-4441
|
| Fax |
773-702-0963
|
| Organization name |
University of Chicago
|
| Department |
Medicine
|
| Lab |
Dolan Lab
|
| Street address |
5841 S. Maryland
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60657 |
| Country |
USA |
| |
|
| Platform ID |
GPL5175 |
| Series (2) |
| GSE7761 |
Identification of Genetic Variants Contributing to Cisplatin-Induced Cytotoxicity using a Genome-wide Approach |
| GSE7851 |
Evaluation of Genetic Variation Contributing to Differences in Gene Expression between Populations |
|
| Relations |
| Alternative to |
GSM189025 |
| Alternative to |
GSM245652 |
