|
Status |
Public on Mar 15, 2012 |
Title |
Adult male HVC replicate 1 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
HVC
|
Organism |
Taeniopygia guttata |
Characteristics |
brain region: HVC gender: Male age: Adult
|
Treatment protocol |
Brain regions HVC and Shelf were laser-capture microdissected from brain sections of adult male zebra finches.
|
Extracted molecule |
total RNA |
Extraction protocol |
Experimental samples: Total RNA was extracted using Absolutely RNA Microprep kit (Stratagene). 200ng total RNA was double-amplified using MessageAmp II aRNA kit (Ambion). 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 |
brain region: Telencephalon gender: Male and female age: Adult
|
Treatment protocol |
Brain regions HVC and Shelf were laser-capture microdissected from brain sections of adult male zebra finches.
|
Extracted molecule |
total RNA |
Extraction protocol |
Experimental samples: Total RNA was extracted using Absolutely RNA Microprep kit (Stratagene). 200ng total RNA was double-amplified using MessageAmp II aRNA kit (Ambion). 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.
|
Description |
ref.HVC.3
|
Data processing |
no data processing (providing only raw data)
|
|
|
Submission date |
Mar 05, 2012 |
Last update date |
Mar 23, 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) |
GSE36270 |
Birdsong "Transcriptomics": Neurochemical Specializations of the Oscine Song System |
|
Relations |
Reanalyzed by |
GSM900020 |