|
| Status |
Public on Jun 02, 2011 |
| Title |
Ball milled aspen rep2 34965202_532 |
| Sample type |
RNA |
| |
|
| Source name |
mycelium, ball-milled aspen
|
| Organism |
Phanerodontia chrysosporium |
| Characteristics |
strain: RP-78
tissue: mycelium
condition: ball-milled aspen
|
| Growth protocol |
5 days growth in shake flasks at 37C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 200 units of SuperScript II (Invitrogen) for 60 min at 42°C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07 units per ul DNA ligase (Invitrogen), 0.27 units per ul DNA polymerase I (Invitrogen), 0.013 units per ul RNase H (Invitrogen), at 16°C for 2 hours. Immediately following, 10 units T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16°C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37°C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water.
|
| Label |
Cy3
|
| Label protocol |
One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37°C.
|
| |
|
| Hybridization protocol |
Hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42°C.
|
| Scan protocol |
Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4.
|
| Description |
34965202_532
|
| Data processing |
Quantile normalization and robust multi-array averaging (RMA) were applied using the DNASTAR Arraystar module (version 4).
|
| |
|
| Submission date |
Mar 14, 2011 |
| Last update date |
Jun 02, 2011 |
| Contact name |
Dan Cullen |
| E-mail(s) |
dcullen@wisc.edu
|
| Phone |
608-231-9468
|
| Organization name |
UW/FPL
|
| Street address |
One Gifford Pinchot Dr
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
| |
|
| Platform ID |
GPL8022 |
| Series (2) |
| GSE27941 |
Lignocellulose-induced regulation of Phanaerochaete chrysosporium genes |
| GSE29659 |
Phanaerochaete chrysosporium and Postia placenta gene expression in ball-milled aspen or ball-milled pine medium |
|
