|
| Status |
Public on Dec 09, 2008 |
| Title |
HEK293Tcells |
| Sample type |
RNA |
| |
|
| Source name |
human embryonic kidney 293T cell line
|
| Organism |
Homo sapiens |
| Characteristics |
human embryonic kidney 293T cell line
|
| Growth protocol |
HEK 293T cells were cultured in parallel in 2*150 cm3 flasks with DMEM (penicillinstreptomycin) (Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Biochrom) and Glutamax (1x) (Gibco). At passage 20 (confluent state), cells were trypsinized, washed off in PBS, pelleted and resuspended in 2 ml lysis solution RLT (Qiagen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was then extracted from ~ 20 x 10^6 cells per sample (2*HEK samples) using RNeasy Midi extraction kit (Qiagen) by following the manufacturer s instructions. DNA was removed using the on column digestion protocol of the RNeasy Midi extraction kit (Qiagen). Total RNA quality was assessed by spectrophotometry (Nanodrop) and gel electrophoresis (1% agarose).
|
| Label |
biotin
|
| Label protocol |
cDNA labeling and microarray hybridization were performed as described in GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual, following manufacturer’s instructions (P/N 701880 Rev.4, www.affymetrix.com).
|
| |
|
| Hybridization protocol |
All probes were hybridized onto the Human Exon 1.0 ST Array (Affymetrix) following the manufacturer's instructions
|
| Scan protocol |
Microarrays were scanned with the GeneChip® Scanner 3000 7G (Affymetrix)
|
| Description |
A model based analysis for tiling arrays was applied to perform the intra-chip normalization, with the adjustment for exon arrays described by Kapur and colleagues . Quantile normalization was then applied between arrays with Affy package in BioConductor (version 2.2) (R statistical environment, v.2.7.0) .
|
| Data processing |
The probe to exon assignment was done using a chip description file (HsEx10stv2_Hs_ENSE), based on Ensembl v.46 and provided as R package . Exon and gene expression were defined as the mean over probe intensities for both replicates.
|
| |
|
| Submission date |
Nov 05, 2008 |
| Last update date |
Nov 12, 2008 |
| Contact name |
Marc Sultan |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Planck Institute for molecular genetics
|
| Street address |
Ihnestr. 73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7557 |
| Series (1) |
| GSE13474 |
Exon array analysis of two human cell lines (HEK293T Ramos B cell) |
|
