|
| Status |
Public on Feb 01, 2010 |
| Title |
dikaryon vs monokaryon replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. cinerea vegetative monokaryotic mycelia
|
| Organism |
Coprinopsis cinerea |
| Characteristics |
genotype: Strain Okayama7
|
| Growth protocol |
For each biological replicate, monokaryon C. cinerea wild-type strain OK7 and dikaryotic strain OK7xJava6 were gown in liquid YMG culture for 2 days, then harvested tohrough a Buchner funnel and frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from all the tissue collected using RNeasy Plant mini-prep kit.
|
| Label |
Alexa fluor 647
|
| Label protocol |
20ug of total RNA were reverse transcribed and labelled with Alexa fluor using the Invitrogen Superscript Indirect labeling kit.
|
| |
|
| Channel 2 |
| Source name |
C. cinerea vegetative dikaryotic mycelia
|
| Organism |
Coprinopsis cinerea |
| Characteristics |
genotype: Strain Java6 crossed with Okayama7
|
| Growth protocol |
For each biological replicate, monokaryon C. cinerea wild-type strain OK7 and dikaryotic strain OK7xJava6 were gown in liquid YMG culture for 2 days, then harvested tohrough a Buchner funnel and frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from all the tissue collected using RNeasy Plant mini-prep kit.
|
| Label |
Alexa fluor 555
|
| Label protocol |
20ug of total RNA were reverse transcribed and labelled with Alexa fluor using the Invitrogen Superscript Indirect labeling kit.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were blocked prior to hybridisation with 5xSSC, 0.1%SDS, 0.1mg.ml-1 BSA at 42oC for 45 min, after which slides were rinsed twice in room temperature 0.1xSSC for 5 min each, followed by a final 10s rinse in water. Slides were dried by centrifugation for 1min in a Labnet C1303 slide spinner. Arrays were placed in Corning hybridization chambers and mSeries Lifterslips (Erie Scientific) placed over the array grids. Labelled cDNAs were combined in 50μl with 30% deionized formamide (Ambion), 5xSSPE, 0.2% SDS, 1μg.μl-1 tRNA (Invitrogen), 40ng.μl-1 oligo dA (Invitrogen). The hybridization mixture was heated at 100oC for 2min, cooled to 25oC, and applied to the microarray slide. Slides were hybridised for 16 hours at 42oC, then washed for 5 min each in 1xSSC, 0.03%SDS (37oC), then 0.2xSSC (ambient temperature), and finally 0.1xSSC (ambient temperature). Slides were dried by centrifugation, as previously, and scanned immediately.
|
| Scan protocol |
Microarray slides were scanned using a GenePix 4200A scanner (Molecular Devices). PMT settings were adjusted so the dye channels were balanced, with a few saturated spots, to maximize data capture. Spots were identified using GenePix Pro software (Molecular Devices) and manual inspection. Scans were quality assessed using the Basic Hybridization Analysis R script (http://cgb.indiana.edu/downloads/1). Spots were manually flagged to be excluded from analysis if there were areas of poor quality such as scratches or dust. Spots were also flagged for omission, using GenePix software, if they fulfilled any of the following criteria: manually flagged, buffer only spots, spots not found, percentage of saturated pixels in both channels > 3, percentage of pixels above background plus 1 standard deviation in both channels < 60, spot pixels < 40.
|
| Description |
Biological replicate 2 of 3
|
| Data processing |
Data normalization and filtering were performed using the Bioconductor package (http://www.bioconductor.org/) within the R statistical framework (http://www.r-project.org/). OLIN [34] was used for intra-slide normalization and log2 transformation. Data were included in further analysis if two of the four replicates for every timepoint were not flagged, or if all four of the replicates for any individual timepoint were not flagged. Ratio values were exported for further analysis, with individual flagged ratio values omitted. Statistical significance of changing expression was assessed using the one class timecourse function in Significance Analysis of Microarrays (SAM) software
|
| |
|
| Submission date |
Oct 13, 2009 |
| Last update date |
Nov 21, 2009 |
| Contact name |
Claire Burns |
| E-mail(s) |
burnsc@indiana.edu
|
| Organization name |
Indiana University
|
| Department |
Biology
|
| Lab |
Zolan Lab
|
| Street address |
1001 E 3rd St
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47404 |
| Country |
USA |
| |
|
| Platform ID |
GPL7697 |
| Series (1) |
| GSE18540 |
Comparison of C. cinerea vegetative dikaryotic to vegetative monokaryotic mycelia |
|
