|
| Status |
Public on Sep 19, 2011 |
| Title |
P493-6 tet 48hr biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
human cell line tet treated 48 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: P493-6 cells
treatment: 48hrs with tet
|
| Treatment protocol |
Treated cells were incubated with 0.1 ug/ml of tetracycline for 48 hrs
|
| Growth protocol |
P493-6 cells were grown in RPMI medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from both untreated and tet treated cells were extracted using a Qiagen RNeasy kit.
|
| Label |
Biotin
|
| Label protocol |
RNA samples were labeled using the Whole Transcript Sense Target Labeling protocol described by Affymetrix and reagent from Affymetrix. Briefly, ribosomal RNA was reduced from the samples using Ribominus Human/Mouse Transcriptome isolation kit (Invitrogen, Carlsbad, California) from 1 micrograms of total RNA. The isolated RNA was then used to synthesize first strand cDNA using random oligonucleotides with T7 promoter as primer and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, the product was purified using cDNA clean-up column, and cRNA was generated through in vitro transcription. 10 ug of cRNA was then used to generate sense strand cDNA using random primer, dNTP-dUTP mix, and Superscript II reverse transcriptase (Invitrogen, Carlsbad, California). Sense strand cDNA was then purified using cDNA clean-up column, fragmented using UDG and APE at 37C for 60 minutes, and terminal labeled with biotinylated nucleotide and terminal DNA transferase at 37C for 60 minutes
|
| |
|
| Hybridization protocol |
The labeled sense strand DNA was hybridized to the Affymetrix GeneChip human Exon 1.0 ST arrays for 17hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cDNA. The staining was further amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate.
|
| Scan protocol |
Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was perfomed through the Affymetrix GeneChip Command Console version 2.0 (AGCC v2.0) software from Affymetrix, using the standard default settings.
|
| Description |
Human P493-6 cell line tet treated 48hr
|
| Data processing |
Data were processed using GeneBASE software. MAT background correction were applied to probe intensities, gene level expression were summarized from the corrected intensities and then quantile normalized.
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.comprehensive.mps
|
| |
|
| Submission date |
Sep 19, 2011 |
| Last update date |
Sep 19, 2011 |
| Contact name |
Hongkai Ji |
| E-mail(s) |
hji@jhsph.edu
|
| Organization name |
Johns Hopkins Bloomberg School of Public Health
|
| Department |
Department of Biostatistics
|
| Street address |
615 North Wolfe Street, Rm E3638
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL5175 |
| Series (2) |
| GSE32219 |
Gene expression from human B-lymphocytes (P493-6) |
| GSE32220 |
Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation |
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