|
| Status |
Public on Dec 22, 2010 |
| Title |
Ind-8_Zn_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Ind-8 cells grown in ZnCl2 supplemented medium
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Ind-8
|
| Treatment protocol |
16 - 24 hrs later, the culture medium was replaced with media containing 65 uM ZnCl2. Cells were harvested by trypsinization 48 hours later (i.e., two cell cycles).
|
| Growth protocol |
Cells were grown for 3 days in Zn-free medium to about 50% confluency, trypsinized, and plated in Zn-free medium (DMEM, 10% dialyzed fetal bovine serum, 5 ug/ml puromycin, with the addition of 1 ug/ml G418 for tI-3 cells) at a cell number:plating area:medium volume ratio of 100,000 cells:75 cm2:20 ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol, followed by Qiagen Rneasy. Only cultures meeting strict quality control standards (population division cycles = 1.8-2.2) for ideal symmetric self-renewal versus asymmetric self-renewal, respectively, were used.
|
| Label |
biotin
|
| Label protocol |
Five micrograms of total RNA was used to make cDNA, and the cDNA was used to make biotinylated cRNA.
|
| |
|
| Hybridization protocol |
cRNA was fragmented and hybridized onto the Affymetrix mouse whole genome GeneChip® 430 2.0 array. The arrays were washed and quantified with a fluorescence array scanner.
|
| Scan protocol |
Quantification and statistics were performed using model-based expression and the perfect match (PM) minus mismatch (MM) method in the G-COS® software (version 1.0) and/or the dChip software version 2005. Data across 8 arrays were originally normalized by setting target intensity at 500 in the G-COS.
|
| Description |
Ind-8 is a p53-inducible cell line that expresses physiological levels of wild-type mouse p53 in response to Zn.
|
| Data processing |
Additional analysis of the data was performed in R/Bioconductor, using .CEL files and the affy() package. Background correction, normalization, and summarization was carried out using RMA. A custom chip description file (Mouse4302_Mm_REFSEQ) which organizes the probes based on RefSeq was used. The ID_REF values in the attached matrix are the probeset names from the custom CDF, and except for control probesets consist of a refseq ID with an _at suffix.
|
| |
|
| Submission date |
Nov 15, 2010 |
| Last update date |
Dec 22, 2010 |
| Contact name |
James Sherley |
| E-mail(s) |
janet.l.smith@gmail.com, sherleyj@bbri.org
|
| Organization name |
Boston Biomedical Research Institute
|
| Department |
Programs in Regenerative Biology and Cancer Biology
|
| Lab |
Adult Stem Cell Technology Center
|
| Street address |
64 Grove St.
|
| City |
Watertown |
| State/province |
MA |
| ZIP/Postal code |
02472 |
| Country |
USA |
| |
|
| Platform ID |
GPL10773 |
| Series (1) |
| GSE25334 |
Asymmetric self-renewal associated (ASRA) genes |
|
