|
| Status |
Public on Jul 01, 2009 |
| Title |
Meiosis 1.5 hours Replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
FAIRE Enriched DNA from cells 15hr after transfer to sporulation media (SM)
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SHy002
|
| Growth protocol |
Synchronous sporulation was carried out by using YPD overnight cultures of wild-type cells to inoculate YPA (1% yeast extract, 2% peptone, 2% potassium acetate) cultures at an OD600 of 0.1. YPA cultures were grown at 30C to an OD600 of 0.8-1.2 (about 16 hours). Cells were collected by centrifugation and washed with SM (2% potassium acetate). The cells were resuspended at an OD600 of 2.0 in SM (~600ml) and incubated in 2L flasks at 30C with shaking at 265 rpm.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
50 ml of cells were fixed with 1% formaldehyde for 20 minutes at room temperature and washed twice with 1X cold PBS. The samples were then snap frozen in a dry ice/ethanol bath and stored at -80C. FAIRE was carried out as described (Hogan, Lee and Lieb 2006). Briefly, cells were thawed and lysed with glass beads and the chromatin was sheared to an average size of ~800 bp by sonication as measured by agarose gel electrophoresis. We conducted a phenol-chloroform extraction on the crosslinked samples (1 mg of extract) to remove proteins and protein-associated DNA. The resulting DNA samples were further purified using Zymo Clean and Concentrator kit.
|
| Label |
Cy3
|
| Label protocol |
~100 ng starting material was amplified using the Whole-Genome Amplification kit (Sigma) as described (O'Geen, Nicolet, Blahnik, Green and Farnham 2006). Two micrograms of amplified DNA was labeled with Cy3 conjugated dUTP using the BioPrime Array CGH kit (Invitrogen). DNA concentration and dye incorporation were measured using a Nanodrop spectrometer.
|
| |
|
| Channel 2 |
| Source name |
Genomic DNA prepped from unfixed (no Formaldehyde) sample
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SHy002
|
| Growth protocol |
Vegetative cells were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
100 ml of UNFIXED cells were washed twice with 1X cold PBS. The cells were lysed with glass beads and the chromatin was sheared to an average size of ~800 bp by sonication as measured by agarose gel electrophoresis. We conducted a phenol-chloroform extraction on the UNFIXED samples to remove proteins. The resulting sonicated genomic DNA was precipitated with isopropanol and resuspended in 10 mM Tris-Cl pH 7.4.
|
| Label |
Cy5
|
| Label protocol |
~100 ng starting material was amplified using the Whole-Genome Amplification kit (Sigma) as described (O'Geen, Nicolet, Blahnik, Green and Farnham 2006). Two micrograms of amplified DNA was labeled with Cy5 conjugated dUTP using the BioPrime Array CGH kit (Invitrogen). DNA concentration and dye incorporation were measured using a Nanodrop spectrometer.
|
| |
|
| |
| Hybridization protocol |
Four micrograms of fluorescently labeled reference DNA and four micrograms of fluorescently labeled FAIRE DNA were used in the hybridization. Samples were competitively hybridized with the reference at 52C for 16 hours in a Maui hybridization chamber to yeast whole genome 4 x 44k tiling microarrays (Agilent; ~270 bp resolution).
|
| Scan protocol |
The arrays were scanned with an Axon 4000B scanner, and data was extracted using GenePix 6.0 software.
|
| Description |
FAIRE Meiosis Time Course - Meiosis 1.5 hours Replicate 2
|
| Data processing |
Probes of poor quality by visual inspection, with more than 10% saturated pixels in either channel, or with a background corrected sum of medians for both channels less than 500, were excluded from analysis (flagged as bad in GenePix). Additionally, those probes representing the mitochondrial genome were excluded. For the remaining probes, the median of the ratios (Cy5/Cy3) for each probe was converted to a log2 ratio. These log2 ratios (Cy5/Cy3) were then converted to z scores by centering at a mean of zero and scaling the standard deviation to one.
|
| |
|
| Submission date |
May 19, 2009 |
| Last update date |
May 21, 2009 |
| Contact name |
Sean Erik Hanlon |
| E-mail(s) |
sehanlon@gmail.com
|
| Phone |
919-843-3229
|
| Organization name |
University of Chicago
|
| Department |
Department of Human Genetics
|
| Lab |
Jason Lieb
|
| Street address |
920 E. 58th Street – CLSC 515E
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL4131 |
| Series (2) |
| GSE16163 |
Meiotic time course of open chromatin as measured by FAIRE |
| GSE18256 |
Meiotic time course: open chromatin and expression profile |
|
