|
| Status |
Public on Mar 23, 2009 |
| Title |
GM19103 |
| Sample type |
RNA |
| |
|
| Source name |
lymphoblastoid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
Coriell cell line repository identifier: GM19103
http://locus.umdnj.edu/nigms/nigms_cgi/display.cgi?GM19103
Yoruban sample
Gender: Male
Associated family: Y042-1
Family relationship: child
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM19103
|
| Treatment protocol |
Each sample was collected while in exponential growth, all remained untreated. Baseline expression data.
|
| Growth protocol |
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 37° C 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 1ug 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 45° C for 16 hours per manufacturer's 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 |
Homo sapiens lymphoblastoid cell lines (30 CEPH trios and 30 Yoruban trios) were purchased from Coriell Institute for Medical Reseach (Camden, NJ).
|
| Data processing |
Expression arrays were analyzed using the Affymetrix PowerTools v1.8.6 (http://www.affymetrix.com/support/developer/powertools/index.affx). The start and end coordinates of all probes represented on the exon array were queried and determined against the human genome (hg18). The coordinates for all SNPs were then queried in the dbSNP database (release 129) (http://www.ncbi.nlm.nih.gov/projects/SNP) and used to identify probes harboring SNPs. Of the ~1.4 million probesets on the exon array, ~288,400 contained at least one probe with a SNP. The probeset signal intensity files were filtered by removing those ~288,400 probes from the probesets harboring these known SNPs. Probe intensities were then background corrected and quantile normalized over all 176 samples. The data were then log2 transformed with a median polish. Gene-level expression of 17,879 transcript clusters was summarized using the RMA (robust multi-array average) method with signals generated on a core set (i.e. with RefSeq-supported annotation) of exons (~288,400 probesets). A transcript cluster or probeset was defined to be reliably expressed in LCLs if the log2 transformed expression signal was greater than 5 in at least 80% of the 176 samples. 10,796 of the 17,879 core transcript clusters met these criteria(15). To avoid annotation ambiguity, the final analysis dataset is comprised of 7,860 expressed transcript clusters (corresponding to 115,544 probesets, a minimum of 3 probesets for each transcript cluster) with unique gene annotations (based on NCBI Human Genome Build 34) as retrieved at the Affymetrix NetAffx Ananlysis Center (http://www.affymetrix.com/analysis/index.affx).
|
| |
|
| Submission date |
Nov 28, 2007 |
| Last update date |
Aug 14, 2011 |
| 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 |
GPL5188 |
| Series (1) |
| GSE9703 |
Identification of Common Genetic Variants that Account for Transcript Isoform Variation between Human Populations |
|
| Relations |
| Alternative to |
GSM188719 |
| Alternative to |
GSM188901 |
