|
| Status |
Public on Dec 13, 2017 |
| Title |
S.pombe-rpb1-CTD-S2A / White-rep2 |
| Sample type |
RNA |
| |
|
| Source name |
rpb1-CTD-S2A / White at exponentially-growing phase replicate 2
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
medium: YES
temperature: 30 C
genotype/variation: rpb1
colony color: white
|
| 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 5x106 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%).
|
| 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.
|
| |
|
| Submission date |
Dec 20, 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) |
| GSE53568 |
Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi |
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