|
| Status |
Public on Jun 27, 2011 |
| Title |
Pyrococcus furiosus DSM 3638 growth curve. Array 252330910012 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pyrococcus furiosus DSM 3638 [reference sample]
|
| Organism |
Pyrococcus furiosus DSM 3638 |
| Characteristics |
growth phase: mid-log phase (OD600=0.096)
|
| Biomaterial provider |
Michael W.W. Adams, Univ. Georgia, Athens, GA
|
| Treatment protocol |
Culture samples were removed anaerobically using a sterile syringe and transfered into sterile 50 ml falcon tubes placed in RT water. Samples were spun at ~300 xg for 25 min (18°C). Spent media was removed and cell pellet was flash frozen in liquid nitrogen before storing at -80°C.
|
| Growth protocol |
Cells were cultured in standard Pf base media supplemented with 0.1% w/v yeast extract (DIFCO technical grade) and 0.3% w/v maltose (Sigma M2250).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using mirVana miRNA isolation kit (Applied Biosystems, Austin, TX) following manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
1.5 µg of total RNA were directly labeled with 1.5 µl of Kreatech dye (Cy3 or Cy5) at 95°C for 5 min, then put it on ice. The solutions were purified by KREApure column.
|
| |
|
| Channel 2 |
| Source name |
Pyrococcus furiosus DSM 3638 growth curve [test sample]
|
| Organism |
Pyrococcus furiosus DSM 3638 |
| Characteristics |
growth phase: mid-log phase (OD600=0.041)
time: 6.5h
replicate: 2
|
| Biomaterial provider |
Michael W.W. Adams, Univ. Georgia, Athens, GA
|
| Treatment protocol |
Culture samples were removed anaerobically using a sterile syringe and transfered into sterile 50 ml falcon tubes placed in RT water. Samples were spun at ~300 xg for 25 min (18°C). Spent media was removed and cell pellet was flash frozen in liquid nitrogen before storing at -80°C.
|
| Growth protocol |
Cells were cultured in standard Pf base media supplemented with 0.1% w/v yeast extract (DIFCO technical grade) and 0.3% w/v maltose (Sigma M2250).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using mirVana miRNA isolation kit (Applied Biosystems, Austin, TX) following manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
1.5 µg of total RNA were directly labeled with 1.5 µl of Kreatech dye (Cy3 or Cy5) at 95°C for 5 min, then put it on ice. The solutions were purified by KREApure column.
|
| |
|
| |
| Hybridization protocol |
7.5 µg of labeled sample RNA and 7.5 µg of labeled reference RNA were fragmented in total 250 µl mixture (50 µl of 10X blocking agent, 10 µl of 25X fragmentation buffer, H2O) at 60°C for 30 min, then put it on ice for 1 min. 250 µl of fragmentation mix and 250 µl of GE hybridization buffer HI-RPM were mixed and spinned for 1 min at RT at 13,000 rpm, and put on ice. 490 µl of the hybridization mix was applied to the slide in chamber, and put in a hybridization oven at 65°C for 17 hrs. After hybridization, the slide was washed sequentially in GE wash buffer 1 (twice) and 2.
|
| Scan protocol |
The arrays were scanned by ScanArray (Perkin Elmer).
|
| Description |
ch1: Pyrococcus furiosus DSM 3638 at OD600=0.096
ch2: Pyrococcus furiosus DSM 3638 at OD600=0.041, t=6.5h, Replicate 2
|
| Data processing |
Signal intensities and local backgrounds were determined by Feature Extraction software (Agilent Technologies). Cy3 and Cy5 intensities were normalized by scaling so that the 75th percentile in the Cy3 and Cy5 channels were equal.
|
| |
|
| Submission date |
Jan 20, 2011 |
| Last update date |
Jun 27, 2011 |
| Contact name |
Sung Ho Yoon |
| Organization name |
Konkuk University
|
| Department |
Department of Bioscience and Biotechnology
|
| Street address |
120 Neungdong-ro, Gwangjin-gu
|
| City |
Seoul |
| ZIP/Postal code |
05029 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL11622 |
| Series (2) |
| GSE26778 |
Pyrococcus furiosus DSM 3638 growth curve, tiling arrays |
| GSE26782 |
Parallel evolution of transcriptome structure during genome reorganization |
|
