|
| Status |
Public on Jan 01, 2016 |
| Title |
Microaerobic-xylose cy3-2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Microaerobic-xylose cy3
|
| Organism |
Ogataea angusta |
| Characteristics |
strain: DL-1-L
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To grow the cells aerobically, 500 ml Erlenmeyer flasks containing 50 ml of the medium were inoculated at an initial OD600 of 0.3 units with overnight pre-cultures and incubated at 37°C while shaking at 180 rpm in an orbital shaker. For microaerobic growth, 250 ml Erlenmeyer flasks containing 100 ml of the medium were inoculated at an initial OD600 of 0.3 units with overnight pre-cultures and were incubated at 37°C while shaking at 100 rpm in an orbital shaker. Cell growth was monitored by determining the optical density (OD600) with a SHIMADZU UV-2550 Spectrophotometer. The total RNA was extracted by a hot phenol method followed by purification using an RNeasy column kit (Qiagen)
|
| Label |
cy3
|
| Label protocol |
30 µg total RNA of each samples was used for first-strand cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, USA) with the oligo dT primer. The cDNA labeling was performed using an Aminoallyl post-DNA labeling kit (GeneChem, Korea) following the manufacturer’s protocol with some modifications.
|
| |
|
| Channel 2 |
| Source name |
Microaerobic-glucose cy5
|
| Organism |
Ogataea angusta |
| Characteristics |
strain: DL-1-L
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To grow the cells aerobically, 500 ml Erlenmeyer flasks containing 50 ml of the medium were inoculated at an initial OD600 of 0.3 units with overnight pre-cultures and incubated at 37°C while shaking at 180 rpm in an orbital shaker. For microaerobic growth, 250 ml Erlenmeyer flasks containing 100 ml of the medium were inoculated at an initial OD600 of 0.3 units with overnight pre-cultures and were incubated at 37°C while shaking at 100 rpm in an orbital shaker. Cell growth was monitored by determining the optical density (OD600) with a SHIMADZU UV-2550 Spectrophotometer. The total RNA was extracted by a hot phenol method followed by purification using an RNeasy column kit (Qiagen)
|
| Label |
cy5
|
| Label protocol |
30 µg total RNA of each samples was used for first-strand cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, USA) with the oligo dT primer. The cDNA labeling was performed using an Aminoallyl post-DNA labeling kit (GeneChem, Korea) following the manufacturer’s protocol with some modifications.
|
| |
|
| |
| Hybridization protocol |
The fluorescent-labeled cDNA was concentrated under a vacuum and resuspended in 15 µl of preheated hybridization buffer (25% formamide, 5x SSC, 0.1% SDS). The pairs of Cys3 and Cys5-labeled samples were mixed and applied to a post-processing H. polymorpha microarray chip.
|
| Scan protocol |
After incubation in the dark at 42°C for 16 – 18 h, the slide was washed, dried, and scanned for Cy5(635 nm) and Cy3(532 nm) signals using a GenePix4000B scanner (Axon Instruments)
|
| Description |
The transcriptomes of Hansenula polymorpha grown on xylose were compared with those of glucose-grown cells under both aerobic and microaerobic conditions
|
| Data processing |
Spot identification and fluorescent signal quantitation were conducted using GenePix Pro v6.0 software, where any flagged, not found, saturated, or bad signal spot was automatically and manually eliminated.
Data processing and normalization were performed with the Arraynorm program. This data has the Matrix worksheet in your submission contains the normalized data that was generated by the Arraynorm software.
|
| |
|
| Submission date |
Jan 02, 2013 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Oh Cheol Kim |
| E-mail(s) |
kill572@kribb.re.kr
|
| Organization name |
KRIBB
|
| Street address |
Gwahakro 125
|
| City |
Daejeon |
| ZIP/Postal code |
82 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL16437 |
| Series (1) |
| GSE43240 |
Transcriptome analysis of xylose metabolism in the methylotrophic yeast Hansenula polymopha |
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