|
| Status |
Public on Sep 01, 2016 |
| Title |
Rap1-depletion-Expression Meiosis TC - YPA 20hrs Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RT cDNA from YPA 20 hours
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SHy20 (tetO7-TATA-Rap1)
growth condition: YPA 20 hours
treatment status: Minus doxycycline (Rap1 on)
molecule: Reverse transcribed total RNA
|
| Treatment protocol |
0.33 ug/ml doxycycline
|
| Growth protocol |
SHy20 (RAP1promoter::kanRtetO7- TATA/RAP1promoter::kanR-tetO7-TATA URA3::CMV-tetTA/URA3::CMV-tetTA) cells were grown overnight in YPD and then inoculated at an OD600 of 0.1 in YPA (containing 0.33 μg/ml doxycycline). Cells were grown in YPA for 8 hours at 30C and then the culture was split into a minus doxycycline (0.33 μg/ml; doxycycline) and a plus doxycycline sample (50 μg/ml doxycycline). The cells were returned to 30C and grown an additional 12 hours. Cells were harvested by centrifugation and washed with SM (either containing no doxycycline or 50 μg/ml doxycycline) resuspended in SM (either containing no doxycycline or 50 μg/ml doxycycline) at an OD600 of 2.0 in SM and incubated with vigorous shaking at 30C. Cells were collected from both minus and plus doxycycline samples after 20 hours in YPA media (25 ml of cells for ChIP and 15 ml for RNA isolation) and after 0, 3, 6, 9 or 12 hours in SM (11 ml for RNA isolation). Samples were pelleted and snap frozen in a dry ice/ethanol bath and stored at -80C until RNA abundance/expression analysis was performed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen samples using hot acid-phenol as previously described (Ausubel 1997).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (30 micrograms) was reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen) incorporating amino-allyl dUTP (Sigma) at a ratio of 3:2 with dTTP. Reactive Cy5 (Amersham) was coupled to the amino-allyl of the resulting DNA fragments in the presence of sodium bicarbonate.
|
| |
|
| Channel 2 |
| Source name |
RT cDNA from YPA 20 hours; Rap1 depleted
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SHy20 (tetO7-TATA-Rap1)
growth condition: YPA 20 hours
treatment status: Plus doxycycline (Rap1 depleted)
molecule: Reverse transcribed total RNA
|
| Treatment protocol |
50 ug/ml doxycycline
|
| Growth protocol |
SHy20 (RAP1promoter::kanRtetO7- TATA/RAP1promoter::kanR-tetO7-TATA URA3::CMV-tetTA/URA3::CMV-tetTA) cells were grown overnight in YPD and then inoculated at an OD600 of 0.1 in YPA (containing 0.33 μg/ml doxycycline). Cells were grown in YPA for 8 hours at 30C and then the culture was split into a minus doxycycline (0.33 μg/ml; doxycycline) and a plus doxycycline sample (50 μg/ml doxycycline). The cells were returned to 30C and grown an additional 12 hours. Cells were harvested by centrifugation and washed with SM (either containing no doxycycline or 50 μg/ml doxycycline) resuspended in SM (either containing no doxycycline or 50 μg/ml doxycycline) at an OD600 of 2.0 in SM and incubated with vigorous shaking at 30C. Cells were collected from both minus and plus doxycycline samples after 20 hours in YPA media (25 ml of cells for ChIP and 15 ml for RNA isolation) and after 0, 3, 6, 9 or 12 hours in SM (11 ml for RNA isolation). Samples were pelleted and snap frozen in a dry ice/ethanol bath and stored at -80C until RNA abundance/expression analysis was performed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen samples using hot acid-phenol as previously described (Ausubel 1997).
|
| Label |
Cy5
|
| Label protocol |
Total RNA (30 micrograms) was reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen) incorporating amino-allyl dUTP (Sigma) at a ratio of 3:2 with dTTP. Reactive Cy5 (Amersham) was coupled to the amino-allyl of the resulting DNA fragments in the presence of sodium bicarbonate.
|
| |
|
| |
| Hybridization protocol |
Labeled reverse transcribed total RNA from Rap1 depeleted and non-depleted samples at each time point were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb). Hybridizations were carried out overnight at 65 C.
|
| Scan protocol |
The arrays were scanned with an Axon 4000B scanner, and data was extracted using GenePix 6.0 software.
|
| Description |
Comparing RNA expression dynamics during meiosis timecourse in cells with wildtype Rap1 levels and cells depleted of Rap1. YPA (respiratory) growth sample.
|
| Data processing |
Probes of poor quality by visual inspection, with more than 10% saturated pixels in either channel, with a background corrected sum of medians for both channels less than 500, or with fewer than 40 foreground pixels were excluded from analysis (flagged as bad in GenePix). Additionally, failed probes, probes representing intergenic regions, probes representing rRNA, probes representing telomeres and probes representing the mitochondrial genome were excluded. For the remaining probes, the median of the ratios (total RNA; Rap1 depleted/non-depeleted cells) for each probe was converted to a log2 ratio. These total RNA log2 ratios (Rap1 depleted/non-depeleted cells) were then converted to z scores by centering at a mean of zero and scaling the standard deviation to one.
|
| |
|
| Submission date |
Nov 20, 2015 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Sean Erik Hanlon |
| E-mail(s) |
[email protected]
|
| 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 |
GPL4414 |
| Series (2) |
| GSE75259 |
Meiosis timecourse Rap1-depletion expression profile |
| GSE75263 |
Meiosis timecourse Rap1 |
|
