|
| Status |
Public on Dec 26, 2008 |
| Title |
Rsc8 ChIP in a rsc3-1 background |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Rsc8-IP genomic DNA from rsc3-1 Saccharomyces cerevisiae carrying Rsc8-TAP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
BY4741, his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rsc3-1::kanR rsc8-TAP::HIS3MX6
|
| Growth protocol |
Cells were grown at 22°C (permissive) until mid-log phase, then an equal volume of hot medium was added to equilibrate the culture to 37°C (restrictive). After 7 hours of growth at restrictive conditions, formaldehyde was added to a final concentration of 1% and cells were crosslinked for 20 mins.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After formaldehyde crosslinking, cells were homogenized and extracts were sonicated on a Branson Sonifier 450 to shear the chromatin to an average size of ~500 bp. A single pulldown was performed with IgG sepharose beads and following decrosslinking and LM-PCR amplification of purified IP DNA, the sample was labeled and hybridized on a Nimblegen array.
|
| Label |
Cy5
|
| Label protocol |
Cy5 labeled primers and Klenow, as per NimbleGen labeling protocol
|
| |
|
| Channel 2 |
| Source name |
Input genomic DNA from rsc3-1 Saccharomyces cerevisiae carrying Rsc8-TAP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
BY4741, his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rsc3-1::kanR rsc8-TAP::HIS3MX6
|
| Growth protocol |
Cells were grown at 22°C (permissive) until mid-log phase, then an equal volume of hot medium was added to equilibrate the culture to 37°C (restrictive). After 7 hours of growth at restrictive conditions, formaldehyde was added to a final concentration of 1% and cells were crosslinked for 20 mins.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After formaldehyde crosslinking, cells were homogenized and extracts were sonicated on a Branson Sonifier 450 to shear the chromatin to an average size of ~500 bp. A small fraction of the chromatin extract was decrosslinked and following LM-PCR amplification of purified input DNA, the sample was labeled and hybridized on a Nimblegen array.
|
| Label |
Cy3
|
| Label protocol |
Cy3 labeled primers and Klenow, as per NimbleGen labeling protocol
|
| |
|
| |
| Hybridization protocol |
Competitively hybridized 1ug of DNA from channels 1 and 2 against each other on NimbleGEn 385k feature, 32bp whole genome tiling array in a Maui SL Mixer with a Maui hybrdization system overnight at 42C.
|
| Scan protocol |
Array was scanned on GenePix 4000B scanner as per NimbleGen guidelines
|
| Description |
Rsc8-IP vs. Input genomic DNA in a rsc3-1 background
|
| Data processing |
The data was prepared using NimbleScan's GFF generating algorithm from the supplementary PAIR files, the VALUE reported is the normalized log2ratio from NimbleScan. The normalization is the default done by NimbleScan. Specifically, the log2 ratio is computed and scaled to center the ratio data around zero. Scaling is performed by subtracting the bi-weight mean for the log2-ratio values for all features on the array from each log2-ratio value.
|
| |
|
| Submission date |
Nov 26, 2008 |
| Last update date |
Dec 14, 2008 |
| Contact name |
Lourdes Pena-Castillo |
| E-mail(s) |
lourdes.pena@gmail.com
|
| Phone |
416 946-7838
|
| Fax |
416 978-8528
|
| Organization name |
University of Toronto
|
| Department |
Banting and Best Department of Medical Research
|
| Lab |
Hughes Lab
|
| Street address |
160 College St. Room 1350
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7699 |
| Series (1) |
| GSE12349 |
A library of yeast transcription factor motifs |
|
