|
| Status |
Public on Mar 24, 2012 |
| Title |
AL of p20 Novel song replicate 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Novel song
|
| Organism |
Taeniopygia guttata |
| Characteristics |
song exposure: 30 minutes of Novel song
brain region: AL (auditory lobule)
gender: male
age: day 20 post-hatch
|
| Treatment protocol |
Post-hatch day 20 male zebra finches that had been raised in acoustic isolation with a foster female were placed in a song playback chamber. The next day, birds heard either silence (control) or 30 minutes of novel song. Birds were then sacrificed and the AL (auditory lobule) was dissected and flash frozen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental samples: Total RNA was extracted using RNAqueous Mini kit (Ambion) and DNase treated with TURBO DNase (Ambion), and cleaned on a spin column. 500ng total RNA was amplified using Agilent's Low RNA input linear amplification kit. Universal reference: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; equal amounts of total RNA from each bird was pooled; total RNA was amplified from this pool using Agilent's Low RNA input linear amplification kit to create sufficient aRNA for 5000 assays.
|
| Label |
Cy5
|
| Label protocol |
1 µg of aRNA was primed with 3 µl of 100 µM random hexamer primers at 70C for 10 min, then reversed transcribed at 42C for 16 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM dATP, dCTP, dGTP, 60µM dTTP and 40µM aa-dUTP; RNA was hydrolyzed in the presence of NaOH and EDTA and samples were cleaned up on a spin column. Finally, Cy3 and Cy5 dye esters (GE Healthcare) were coupled to aa-dUTP residues in NaCO3 buffer for 2 hrs at room temp, then neutralized and cleaned on spin columns.
|
| |
|
| Channel 2 |
| Source name |
universal SoNG reference
|
| Organism |
Taeniopygia guttata |
| Characteristics |
song exposure: NA
brain region: Telencephalon
gender: Male and female
age: Adult
|
| Treatment protocol |
Post-hatch day 20 male zebra finches that had been raised in acoustic isolation with a foster female were placed in a song playback chamber. The next day, birds heard either silence (control) or 30 minutes of novel song. Birds were then sacrificed and the AL (auditory lobule) was dissected and flash frozen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental samples: Total RNA was extracted using RNAqueous Mini kit (Ambion) and DNase treated with TURBO DNase (Ambion), and cleaned on a spin column. 500ng total RNA was amplified using Agilent's Low RNA input linear amplification kit. Universal reference: Total RNA was extracted using Trizol following manufacturer's protocol; samples were Dnase treated and cleaned on a spin column; equal amounts of total RNA from each bird was pooled; total RNA was amplified from this pool using Agilent's Low RNA input linear amplification kit to create sufficient aRNA for 5000 assays.
|
| Label |
Cy3
|
| Label protocol |
1 µg of aRNA was primed with 3 µl of 100 µM random hexamer primers at 70C for 10 min, then reversed transcribed at 42C for 16 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM dATP, dCTP, dGTP, 60µM dTTP and 40µM aa-dUTP; RNA was hydrolyzed in the presence of NaOH and EDTA and samples were cleaned up on a spin column. Finally, Cy3 and Cy5 dye esters (GE Healthcare) were coupled to aa-dUTP residues in NaCO3 buffer for 2 hrs at room temp, then neutralized and cleaned on spin columns.
|
| |
|
| |
| Hybridization protocol |
Samples were suspended in SlideHyb#1 (Ambion), applied to slides in Corning Hybridization Chambers, and incubated O/N at 42C. After hybridization, the slides were washed sequentially in 1X SSC, 0.2% SDS; 0.1X SSC, 0.2% SDS; and 0.1X SSC for 5 minutes each, and spun dry.
|
| Scan protocol |
Scanned on an Axon GenePix 4000B scanner
Images were quantified using Axon GenePix 6.0.
|
| Data processing |
no data processing (providing only raw data)
|
| |
|
| Submission date |
Mar 21, 2012 |
| Last update date |
Mar 24, 2012 |
| Contact name |
Kirstin Replogle |
| E-mail(s) |
replogle@igb.uiuc.edu
|
| Organization name |
University of Illinois
|
| Street address |
1206 W Gregory Drive
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL9554 |
| Series (1) |
| GSE36657 |
Developmental shifts in gene expression in the auditory forebrain during the sensitive period for song learning |
|
| Relations |
| Reanalyzed by |
GSM900098 |
