|
| Status |
Public on Apr 04, 2013 |
| Title |
Mycelium_CYAs_10d |
| Sample type |
RNA |
| |
|
| Source name |
Fungal mycelium, grown on CYAs medium, cultivated for 10 days
|
| Organism |
Aspergillus nidulans |
| Characteristics |
tissue: mycelium
growth medium: CYAs
sampling time: 10 days
|
| Treatment protocol |
Whole colonies from three-stab agar plates were sampled for transcriptional analysis by scraping the mycelium off the agar with a scalpel and transferring directly into a 50 mL Falcon tube containing app. 15 mL of liquid nitrogen. Care was taken to transfer a minimum of agar to the Falcon tube. The liquid nitrogen was allowed to evaporate before capping the lid and re-cooling the tube in liquid nitrogen before storing the tube at -80 deg C until use for RNA purification.
|
| Growth protocol |
All strains were three-point inoculated on the media and incubated at 32 deg C in darkness for 4, 8 or 10 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA purification, 40-50 mg of frozen mycelium was placed in a 2 ml microcentrifuge tube, and precooled in liquid nitrogen containing three steel balls (two balls with a diameter of 2 mm and one ball with a diameter of 5 mm). The tubes were then shaken in a Retsch Mixer Mill at 5 deg C for 10 min, until the mycelium was ground to a powder. Total RNA was isolated from the powder using the Qiagen RNeasy Mini Kit, according to the protocol for isolation of total RNA from plant and fungi, including the optional use of the QiaShredder column. Quality of the purified RNA was verified using a NanoDrop ND-1000 and an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
150 ng in 1.5 µL total RNA was labeled according to the One-Color Labeling for Expression Analysis, Quick Amp Low Input (QALI) manual version 6.5 May 2010 from Agilent Technologies. Yield and specific activity were determined on a NanoDrop ND-1000 spectrophotometer and verified on a Qubit 2.0 fluorometer (Invitrogen).
|
| |
|
| Hybridization protocol |
1.65 µg labeled cRNA was fragmented at 60 deg C on a heating block and the cRNA was prepared for hybridization according to the QALI protocol. 100 µl sample was loaded on a 4x44 Agilent Gasket Slide situated in an Agilent Technologies hybridization chamber. The 4x44 array was placed on top of the Gasket Slide. The array was hybridized at 65 deg C for 17 hours in an Agilent Technologies Hybridization oven.
|
| Scan protocol |
The array was washed following the QALI protocol and scanned in a G2505C Agilent Technologies Micro Array Scanner according to the same protocol.
|
| Description |
CYAs_10
|
| Data processing |
The raw array signal was processed by first removing the background noise using the normexp method, and the signal between arrays was made comparable using the quantiles normalization method, as implemented in the Limma package (Smyth et al., 2005). Multiple probe signals per gene were summarized into gene-level expression indices using Tukey's median polish, as performed in the last step of the RMA processing method (Irizarry, 2003).
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| |
|
| Submission date |
Aug 08, 2012 |
| Last update date |
Apr 05, 2013 |
| Contact name |
Mikael R Andersen |
| E-mail(s) |
[email protected]
|
| Organization name |
Technical University of Denmark
|
| Department |
BioCentrum-DTU
|
| Lab |
Center for Microbial Biotechnology
|
| Street address |
Søltofts plads, bygn 223
|
| City |
Kgs. Lyngby |
| ZIP/Postal code |
2800 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL15913 |
| Series (1) |
| GSE39993 |
RNA sampling from a chemically diverse set of solid agar cultures of Aspergillus nidulans |
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