|
| Status |
Public on Feb 10, 2011 |
| Title |
Cigarette smoke extract-chronically treated OKF6 cells |
| Sample type |
genomic |
| |
|
| Source name |
OKF6 cells, chronic cigarette extract treatment for 7 months
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OKF6
cell type: oral immortal keratinocyte, treated for 7 months
|
| Treatment protocol |
Research-grade cigarettes, 2R4F, from the Kentucky Tobacco Research and Development Center at the University of Kentucky were smoked to 0.25 cm above the filter. 100% CSE was prepared by bubbling smoke from one cigarette into 1 ml of PBS. Each puff was 2 seconds long at a rate 35ml/second. This extract was then filtered using a .22um filter from BD biosciences filter. Each dilution was done by volume in media. Treatment concentration was .1%.
|
| Growth protocol |
The cell line was expanded and passaged in keratinocyte serum-free medium (Gibco/Invitrogen; 10725-018). This medium was supplemented with BPE (25 ug/ml), epidermal growth factor (0.2 ng/ml), CaCl2(0.4 mM) and 1% penicillin-streptomycin. Both the cells treated with CSE and passaged cells were cultured in 37 °C humidified air incubators with 5% CO2. All cell lines were grown in 35mm dishes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
phenol/chloroform extraction
|
| Label |
biotin
|
| Label protocol |
GeneChip CustomSeq Resequencing Assay Kit
|
| |
|
| Hybridization protocol |
16hrs at 45° C with rotation (60rpm) as described by Affymetrix in their DNA Array Fluidics Station 450 Protocol.
|
| Scan protocol |
Image analysis by GeneChip Operating Software (GCOS) 1.4 at manufacturer's settings.
|
| Description |
Cigarette Smoke Extract-chronically treated OKF6 cells
|
| Data processing |
All CEL files were analyzed in Affy's GeneChip Sequence Analysis Software, GSEQ, using its "Resequencing Algorithm Version 2", for 8 um arrays. The results of this process are saved as CHP files (containing processed base calls). Analysis was conducted using the recommendations from Affy's Mitochondrial Resequencing Array 2.0 Supporting Documentation on their Support page (http://www.affymetrix.com/support/technical/byproduct.affx?product=humitoreseq) following algorithm parameter settings recommendations 3.1:
- Genome Model = Diploid. Selection of the diploid genome model will enable the detection of heteroplasmy.
- Quality Score Threshold (QST) = 3. The QST parameter was selected to provide the highest performance in terms of overall base calling accuracy and call rates.
FASTA files were exported from GSEQ using proceedures fully described in the gseq_user_guide.pdf, from Affy's webpage. In short, Affy's GSEQ Toolbar provides an "Export -> FASTA" function (alternatively the [FASTA] button) with two options.
The "Whole sequence" option was used to create the submitted file named "SDas051408_Whole_Sequence.txt".
The "SNP flanking sequence" option was used to create "SDas051408_FASTA_Export.txt".
The base calls (from .CHP files) are provided in the file named "SDas051408_TableData.txt".
|
| |
|
| Submission date |
Sep 28, 2010 |
| Last update date |
Feb 10, 2011 |
| Contact name |
wenyue sun |
| E-mail(s) |
wsun10@jhmi.edu
|
| Organization name |
Johns Hopkins Medical Institutions
|
| Department |
Otolaryngology Head and Neck Surgery
|
| Street address |
1550 Orleans street, Rm 574A
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL10983 |
| Series (1) |
| GSE24414 |
Mitochondrial mutations in cigarette smoke extract-chronically treated human oral OKF6 cells |
|
