|
| Status |
Public on May 29, 2008 |
| Title |
NA18505_S |
| Sample type |
RNA |
| |
|
| Source name |
Lymphoblastoid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
SAHA drug response: NA
Simva drug response: NA
6MP drug response: NA
5FU drug response: NA
MTX drug response: NA
|
| Growth protocol |
EBV-transformed lymphoblastoid cell lines were acquired from the NHGRI Sample Repository for Human Genetic Research in frozen aliquots. Cells were thawed in 5mL culture medium (RPMI medium 1640 (Invitrogen) supplemented with 10% FetalPlex (Gemini), 2mM L-Glutamine (Invitrogen), and 1x penicillin/streptomycin (Invitrogen)). Cell lines were counted daily using Z2 Coulter Counter (Beckman Coulter) and passaged as needed to maintain a concentration of 2-5 x 1e5 cells/ml at 37C in a 95% humidified 5% CO2 atmosphere. Initially, cells were grown until 5 x 1e5 cells/ml were reached in 50 mL total volume. Then, ten identical aliquots were frozen in 1 mL freezing media containing 50% FetalPlex, 40% RPMI 1640 medium, and 10% DMSO (Sigma) at -80C for 24 hrs and transferred to liquid nitrogen. These aliquots were used to provide biologic replicates for the experiments described below. Aliquots were thawed on experiment day #1 as described above. Cell lines were counted daily and passaged as need to maintain a concentration of 4-8 x 1e5 cells/ml in 10 mL culture medium. On experiment day #7, cells were counted and distributed for use in the various experiments described below. One cc of culture was used for immediate immunophenotyping via FACS and Luminex beads. One cc of culture was used for RNA and DNA extraction using Trizol (Invitrogen) following the manufacturer's protocol. The remaining eight cc of culture were used for drug response assays described below.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All LCLs were cultured in the fashion described above. Prior to the plating of cells for the Drug Response Assay, 5 x 10^5 cells were set aside for RNA extraction. Cells were immediately lysed with Trizol Reagent (Invitrogen). RNA was collected according to the manufacturer instructions.
|
| Label |
biotin
|
| Label protocol |
As per manufactureer's protocol. 1.25 ug total RNA (OD>1.8) was diluted to a total volume of 10uL.
|
| |
|
| Hybridization protocol |
RNA was processed and hybridized onto Affymetrix Human U133A whole genome RNA expression genechip arrays according to the manufacturer’s protocol.
|
| Scan protocol |
As per manufacturer's protocol
|
| Description |
NA18505_S
|
| Data processing |
RMA; Gene expression summary values for the whole dataset were computed by RMA[36,37] and log-transformed. Measurements were successfully obtained for 257 HapMap cell lines in the main experiment, for 64 biological replicates, for 24 cell lines originally thawed at the WTSI, as well as multiple replicates of 5 cell lines derived from chimpanzees. (raw data in “all broad cell line expression data.zip” online)
|
| |
|
| Submission date |
May 28, 2008 |
| Last update date |
May 29, 2008 |
| Contact name |
Roman Yelensky |
| E-mail(s) |
ryelensky@gmail.com
|
| Organization name |
MGH
|
| Street address |
Massachusetts General Hospital Department of Molecular Biology 185 Cambridge St., Simches Ctr., CPZN-6818
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114-2790 |
| Country |
USA |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE11582 |
Genetic Analysis of Human Traits In-Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines |
|
