|
| Status |
Public on Mar 09, 2012 |
| Title |
beta-TC3 cells, 01-hr treatment, 1.3uM, SAHA, Replicate3, Batch_04, HTA6_A09 |
| Sample type |
RNA |
| |
|
| Source name |
beta-TC3 cells
|
| Organism |
Mus musculus |
| Characteristics |
treatment: 1 hr with 1.3uM, SAHA
|
| Treatment protocol |
Compounds dissolved at 1000-fold the profiling concentration in DMSO were diluted 1:10 with media and 10 µl were added per well. At the indicated time point (1, 6, 24 h, respectively), cells were lysed in 900 µl buffer RLT (Qiagen) by shaking for 30s, and lysates were immediately frozen in liquid nitrogen.
|
| Growth protocol |
Cell lines αTC1 and βTC3 were cultured in low-glucose DMEM supplemented by 10% FBS, 10 U/ml penicillin and 50 µg/ml streptomycin. 24 h prior to compound treatment, 3 mio. cells were plated per well in a 6-well plate in 1 ml medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using Qiagen RNeasy kits according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
1 µg of total RNA was used to prepare complementary RNA (cRNA) target with the Genechip® HT One-Cycle cDNA synthesis Kit (Affymetrix 900687) and the GeneChip® HT IVT Labeling Kit (Affymetrix 900688). Total RNA was first reverse transcribed using a T7-Oligo(dT) promoter primer in the first strand cDNA synthesis reaction. Following RNAse H-mediated second strand cDNA synthesis, the double-stranded cDNA was purified and served as a template for an in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for cRNA amplification and biotin labeling.
|
| |
|
| Hybridization protocol |
The biotinylated cRNA targets were normalized, fragmented and hybridized to Affymetrix HT Mouse 430 A peg arrays (Affymetrix 901045). The hybridization and subsequent washing and staining were performed on the Affymetrix GeneChip® Array Station (GCAS) automation platform.
|
| Scan protocol |
The arrays were scanned on the GeneChip® HT Array Plate Scanner (Affymetrix 00-0332).
|
| Description |
beta-TC3 cells
|
| Data processing |
Expression data (in the form of CEL files) from eight 96-well Affymetrix HT_MG-430A arrays was background corrected, normalized and summarized with RMA using GenePattern.
|
| |
|
| Submission date |
Mar 08, 2012 |
| Last update date |
Mar 09, 2012 |
| Contact name |
Paul A Clemons |
| E-mail(s) |
pclemons@broadinstitute.org
|
| Phone |
617-714-7346
|
| Organization name |
Broad Institute of Harvard and MIT
|
| Department |
Chemical Biology Program
|
| Street address |
7 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02143 |
| Country |
USA |
| |
|
| Platform ID |
GPL8759 |
| Series (1) |
| GSE36379 |
Expression data from mouse pancreatic cell lines treated with chromatin-targeting small molecules |
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