|
| Status |
Public on Sep 01, 2016 |
| Title |
Expression Meiosis TC - Meiosis 4.5hrs Replicate 7 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
RT cDNA from Meiosis 4.5 hours
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SK1 yeast strain SHy002
growth condition: Meiosis 4.5 hours
molecule: Reverse transcribed total RNA
|
| Growth protocol |
Vegetative cells were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8 and 25 ml of cells were collected. Synchronous meiosis/sporulation was carried out by using YPD overnight cultures 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. Cells were collected after 8 hours in YPA media (35 ml of cells) and after 0, 1.5, 3, 4.5, 6, 9 or 12 hours in SM (12.5 ml of cells). 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.
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| |
|
| Channel 2 |
| Source name |
Genomic DNA common reference
|
| Organism |
Saccharomyces cerevisiae SK1 |
| Characteristics |
strain: SK1 yeast strain SHy002
growth condition: Meiosis 4.5 hours
molecule: Genomic DNA common reference
|
| Growth protocol |
Vegetative cells were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8 and 25 ml of cells were collected. Synchronous meiosis/sporulation was carried out by using YPD overnight cultures 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. Cells were collected after 8 hours in YPA media (35 ml of cells) and after 0, 1.5, 3, 4.5, 6, 9 or 12 hours in SM (12.5 ml of cells). 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 |
genomic DNA |
| Extraction protocol |
100 ml of vegetative cells (OD600 0.6-0.8) were pelleted and resuspended in 2 ml lysis buffer (10 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% SDS, 2% Triton X-100). The cells were split into 4 tubes (500 ul each) and an equal volume of phenol:chloroform:isoamyl-alcohol was added to each tube. The cells were then lysed with glass beads. The samples were spun down for 5 minutes at top speed and the aqueous layers was transfered to fresh tubes. Nucleic acids were precipitated with 1 ml of 95% ethanol and resuspended in 200 ul of 1X TE + 30 ug RNAse and incubated at 30 C for 30 minutes. 5 M NaCl was added to a final concentration of 500 mM and the samples were precipitated with isopropanol. Pellets were washed 2X with 70% ethanol and dried at room temperature for 30 minutes. The resulting genomic DNA was resuspended in 10 mM Tris-Cl pH 7.4 and pooled and the concentration was determined with a spectrophotometer.
|
| Label |
Cy5
|
| Label protocol |
Purified genomic DNA (4 micrograms) was labeled with Klenow (NEB) incorporating amino-allyl dUTP (Sigma) at a ratio of 3:2 with dTTP. Reactive Cy3 (Amersham) was coupled to the amino-allyl of the resulting DNA fragments in the presence of sodium bicarbonate.
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| |
|
| |
| Hybridization protocol |
Labeled reverse transcribed total RNA samples and genomic DNA references 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 |
RNA expression dynamics during meiosis timecourse. Meiosis (sporulation) 4.5 hour 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/Genomic DNA common reference) for each probe was converted to a log2 ratio. These log2 ratios (total RNA/Genomic DNA common reference) were then converted to z scores by centering at a mean of zero and scaling the standard deviation to one.
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| |
|
| 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) |
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