|
| Status |
Public on Oct 19, 2010 |
| Title |
YPS2079.T218 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
YPS2079
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YPS2079
time (min after alpha-factor release): 218
|
| Treatment protocol |
Upon reaching a culture density of OD600 ≈ 0.25, α-factor mating pheromone was added to a final concentration of 4 μM. Cultures were then incubated ≈75 min. until arrested and synchronized in late G1. The state of synchronization was determined by the appearance of < 10% shmoos and < 10% budding cells, visualized by light microscopy (100×, oil). Cultures were released from arrest by removing α-factor: 2× wash with 4◦C S medium (SD without dextrose) and resuspension of cell pellets with fresh 18◦C SD medium. Approximately 25 ml aliquots of each culture were distributed into 18 flasks and incubated at 18◦C (225 rpm). Incubation of cultures at 18◦C in SD medium more than doubles the CDC-period, allowing a more accurate comparison of measurements across strains by reducing temporal sampling variation.
|
| Growth protocol |
Strains were inoculated from frozen stock and cultured overnight in SD (synthetic dextrose) minimal medium at 30◦C (225 rpm). The next day cultures were diluted into fresh SD.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each frozen cell culture sample using Qiagen’s RNeasy Kit, following manufacturer’s instructions. cDNA was prepared from 15 μg of each RNA sample using SuperScript III reverse transcriptase (Invitrogen) and compared directly to unsynchronized S. cerevisiae cDNA (YPS183 cultured at 30◦C in YPD until reaching OD600 1.1) on 2-channel spotted-oligo glass microarrays in a common reference design.
|
| Label |
635
|
| Label protocol |
Invitrogen AlexaFluor 555 and 647 fluorophores were used to label each cDNA sample.
|
| |
|
| Channel 2 |
| Source name |
YPS183
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YPS183
growth conditions: unsynchronized, log phase, YPD
|
| Treatment protocol |
Upon reaching a culture density of OD600 ≈ 0.25, α-factor mating pheromone was added to a final concentration of 4 μM. Cultures were then incubated ≈75 min. until arrested and synchronized in late G1. The state of synchronization was determined by the appearance of < 10% shmoos and < 10% budding cells, visualized by light microscopy (100×, oil). Cultures were released from arrest by removing α-factor: 2× wash with 4◦C S medium (SD without dextrose) and resuspension of cell pellets with fresh 18◦C SD medium. Approximately 25 ml aliquots of each culture were distributed into 18 flasks and incubated at 18◦C (225 rpm). Incubation of cultures at 18◦C in SD medium more than doubles the CDC-period, allowing a more accurate comparison of measurements across strains by reducing temporal sampling variation.
|
| Growth protocol |
Strains were inoculated from frozen stock and cultured overnight in SD (synthetic dextrose) minimal medium at 30◦C (225 rpm). The next day cultures were diluted into fresh SD.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each frozen cell culture sample using Qiagen’s RNeasy Kit, following manufacturer’s instructions. cDNA was prepared from 15 μg of each RNA sample using SuperScript III reverse transcriptase (Invitrogen) and compared directly to unsynchronized S. cerevisiae cDNA (YPS183 cultured at 30◦C in YPD until reaching OD600 1.1) on 2-channel spotted-oligo glass microarrays in a common reference design.
|
| Label |
532
|
| Label protocol |
Invitrogen AlexaFluor 555 and 647 fluorophores were used to label each cDNA sample.
|
| |
|
| |
| Hybridization protocol |
Hybridized slides were incubated for 24–65 hours at 42◦C. Samples were hybridized to 2 dye-swapped microarrays. Unsynchronized MA line transcriptomes were produced with the same design. Corning UltraGAPS glass slides, spotted with the Operon AROS for Saccharomyces cerevisiae, V1.1, were used for all hybridizations. Each microarray targets 6388 protein-coding genes using 2 replicate spots per oligo, yielding 4 technical expression measurements per gene, strain, and timepoint. Slides were prepared for scanning by serial incubation in wash buffers and dried using both a vacuum and high-purity, filtered N2 gas.
|
| Scan protocol |
Slides were scanned at 10 μM resolution, 100% power, using an Agilent GenePix 4000B scanning confocal laser microscope. Gain was adjusted manually to optimize signal–noise ratios and to balance channel intensity distributions.
|
| Description |
raw data files: MDUser_13615269.gpr, MDUser_13615255.gpr
raw data file dye swap pattern: 0,1
raw data file platform pattern: J.Kim Yeast 2007, J.Kim Yeast 2007
PROTOCOLS web link: http://kim.bio.upenn.edu/software/yeast-cdc/KimLab_yeast_microarray_protocol.pdf
|
| Data processing |
GenePix v6.0 (Agilent) was used for feature identification and subsequent spot quantification per channel. Bad spots were flagged manually for removal from further analysis; otherwise all spots were treated equally in GenePix. Briefly, data were log transformed and normalized for within and between slide normalization, including lowess transformation. Missing data were imputed using a combination of cubic spline interpolation and K-nearest neighbors. Details for data normalization are described in Supplemental materials and methods in the associated manuscript.
Normalized log2 fluorescence intensity of a gene at a particular timepoint of the cell cycle for a particular strain, relative to fluorescence intensity of that gene in a YPD media batch culture of the laboratory strain YPS183 in log phase; values are the average of within-slide (different spots) and between-slide (dye-swap) technical replicate measurements. The complete matrix of normalized data, including imputed expression data for 7 additional Samples (YPS2060.T111, YPS2060.T152, YPS2060.T194, YPS2067.T87, YPS2067.T111, YPS3137.T63, YPS3137.T227) since the overall experiment involves comparison of time series across yeast strains is provided as a supplementary file on the Series record.
|
| |
|
| Submission date |
Oct 18, 2010 |
| Last update date |
Oct 19, 2010 |
| Contact name |
Daniel Francis Simola |
| E-mail(s) |
simola@mail.med.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Cell and Developmental Biology
|
| Lab |
Shelley L. Berger
|
| Street address |
1041 BRB II/III
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL11064 |
| Series (2) |
| GSE24771 |
Heterochronic Evolution Reveals Modular Timing Changes in Budding Yeast Transcriptomes (Natural Strain Timecourse) |
| GSE24772 |
Heterochronic Evolution Reveals Modular Timing Changes in Budding Yeast Transcriptomes |
|
