|
| Status |
Public on Aug 31, 2013 |
| Title |
S.pombe-pmc6D-pink-rep2 |
| Sample type |
RNA |
| |
|
| Source name |
pmc6 deleted_pink colony_exponentially-growing phase
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: pmc6 deleted
medium: YES
growth temperature: 30°C
|
| Treatment protocol |
A single colony of S. pombe cells on a YES plate was inoculated into YES liquid medium. Cells were incubated at 30°C and collected with filtration when they reached a density of 5x10^6 cells/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated as follows: the cell pellet was frozen with liquid nitrogen and thawed on ice. Cells were washed once with pre-chilled water. The cell pellet was resuspended in 720 micro-l of TES(10mM Tris pH7.5, 10mM EDTA pH8, 0.5% SDS) on ice. 720 micro-l of acidic phenol-chloroform was added to the cell suspension and mixed immediately. The sample was incubated at 65°C for 1 hour, and then placed on ice for 1 min. The sample was centrifuged for 15 min. at 14000rpm at 4°C. 700 micro-l of water phase of the sample was added to 700 micro-l of acidic phenol-chloroform prepared in a phase lock tube (MaXtractTM HighDensity 2ml Qiagen), then mixed. The sample was centrifuged for 5min at 14000rpm at 4°C. 700 micro-l of water phase of the sample was added to 700 micro-l of chloroform prepared in a new phase lock tube, then mixed. The tube was centrifuged for 5 min at 14000rpm at 4°C. Total RNA from the 500 micro-l of the water phase was purified by ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit, one-color including Cy3-CTP (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
50 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/micro-g cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 micro-l containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 micro-l of 2x GE hybridization buffer HI-RPM (Agilent) was added to the fragmentation mixture and hybridized to Agilent custom microarrays (8 x 15k format) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8 x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5 micro-m, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
SAMPLE 10
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (ver 10.7.3.1, Agilent) using default parameters (protocol GE1-107_Sep09 and Grid: 028936_D_F_20100615) to obtain background subtracted and spatially detrended Processed Signal intensities.
5589 probes (including 60 control probes) are spotted multiply to fill 15744 spots in each array. A geometric mean of the gProcessedsignals of the spots for each probe was calculated to make a data set for 5589 probes. The geometric mean was normalized by the 75 percentile method (the value at 75% was set to 2500.) to get the value in the Matrix sheet. ID_REF in the Matrix sheet match the identifiers in the SPOT ID column in the 028936_D_GEO_20110527.txt file. When the average of the gIsWellAboveBG of the spots for each probe is higher than 0.55, the probe signal is considered more than the detection limit, ranked with “a” in the Rank columns.
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| |
|
| Submission date |
Jan 16, 2013 |
| Last update date |
Jan 23, 2023 |
| Contact name |
Atsushi Matsuda |
| Organization name |
National Institute of Information and Communications Technology
|
| Street address |
588-2, Iwaoka, Iwaoka-cho, Nishi-ku
|
| City |
Kobe |
| ZIP/Postal code |
651-2492 |
| Country |
Japan |
| |
|
| Platform ID |
GPL15360 |
| Series (1) |
| GSE43543 |
Mediator directs co-transcriptional heterochromatin assembly by RNAi-dependent and -independent pathways. |
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