|
| Status |
Public on Feb 26, 2008 |
| Title |
FACS-sorted cells with photoconverted Kaede vs. FACS-sorted cells with non-photoconverted Kaede_ZF1351 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FACS-sorted cells with photoconverted Kaede
|
| Organism |
Danio rerio |
| Characteristics |
Zebrafish embryos
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol/Chloroform method according to manifecturer protocol and the RNA is further purified by the Qiagen RNAeazy columns
|
| Label |
Cy5
|
| Label protocol |
0.5-1.0 microgram of total RNA was reverse transcribed with T7 Oligo-dT, followed by a second strand synthesis and overhight i9n vitro transcription in the presence of Aminoallyl UTPs using the Amino Allyl MessageAmpª II aRNA Amplification kit from Ambion (Kit Cat#1753). The Aminoallyl UTP labeled cRNAs are then coupled with either Cy3 or Cy5 mono NHS Ester Dyes. The CyDyes labeled cRNAs are then purified with columns and reagents provided by the Kit (Cat#1753).
|
| |
|
| Channel 2 |
| Source name |
FACS-sorted cells with non-photoconverted Kaede,
|
| Organism |
Danio rerio |
| Characteristics |
Zebrafish embryos
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol/Chloroform method according to manifecturer protocol and the RNA is further purified by the Qiagen RNAeazy columns
|
| Label |
Cy3
|
| Label protocol |
0.5-1.0 microgram of total RNA was reverse transcribed with T7 Oligo-dT, followed by a second strand synthesis and overhight i9n vitro transcription in the presence of Aminoallyl UTPs using the Amino Allyl MessageAmpª II aRNA Amplification kit from Ambion (Kit Cat#1753). The Aminoallyl UTP labeled cRNAs are then coupled with either Cy3 or Cy5 mono NHS Ester Dyes. The CyDyes labeled cRNAs are then purified with columns and reagents provided by the Kit (Cat#1753).
|
| |
|
| |
| Hybridization protocol |
200 picomoles of Cy3 and 100 picomoles of Cy5 labeled cRNAs are put in a total volume of 24 ul (qsp with DEPC H20) and 6 microliter of 5x fragmentation buffer from Affymetrix. the mixture is incubated at 94 ¡C for 15 minutes. Then, 25 ul of 2X hybridization buffer (250 _l Formamide, 250 _l 20xSSC and 20 _l 10%SDS). The mixture is then denatured for 2 minutes and applied on the Maui (BioMicro Systems, Inc) hybridization chamber/ array slide sandwich. The hybridization is performed at 45 degre for 20 hours.
|
| Scan protocol |
Agilent Scanner
|
| Description |
Zebrafish embryos
|
| Data processing |
IPLab/DeArray
|
| |
|
| Submission date |
Feb 26, 2007 |
| Last update date |
Aug 08, 2007 |
| Contact name |
Christopher Pan |
| E-mail(s) |
cpan@mail.nih.gov
|
| Organization name |
NIH
|
| Department |
NHGRI
|
| Street address |
Rm 5228, Bldg 50, South Dr.
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL4609 |
| Series (1) |
| GSE8654 |
Identification of mesendoderm precursor- and neurectoderm precursor-enriched genes in Zebrafish embryos |
|
