|
| Status |
Public on Aug 30, 2007 |
| Title |
IU/Hahn_Drosophila_v2.0 |
| Sample type |
RNA |
| |
|
| Source name |
D. melanogaster embryo 5 hours
|
| Organism |
Drosophila melanogaster |
| Characteristics |
30-50 embryos from line 17
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two RNA samples per line per time point (5 and 8 hours) were extracted using manufacturer's TRIzol reagent protocol (Invitrogen, Carlsbad, CA). The concentrations of these samples were tested using a spectrophotometer and Na2HPO4 spec solution. The 36 samples (9 lines x 2 time points x 2 samples) of extracted RNA were labeled using the one-cycle cDNA Synthesis protocol from Affymetrix. cDNA was made from the extracted RNA by first making a T7-Oligo(dT) Primer Master Mix and letting it incubate with the samples for 10 min at 700C and cooling for 2min at 40C. A First-Strand Master Mix was added and the samples were incubated for 2 min at 420C. After 200U/μL SuperScript II was added the samples were incubated for an additional hour at 420C and then cooled at 40C for 2min. A Second-Strand Master Mix was added to the samples and then they were incubated at 160C for 2 hours and then cooled for 2min at 40C. The samples were incubated for another 5min at 160C after T4 DNA Polymerase was added, the samples were cooled at 40C for 2min and 0.5M EDTA was added. The now double stranded cDNA was cleaned up using spin columns and 100% Ethanol.
|
| Label |
biotin
|
| Label protocol |
An IVT Reaction mix was added to the samples to synthesis Biotin-Labeled cRNA, and they were allowed to incubate at 370C overnight (~16 hours). The newly Biotin-labeled cRNA was cleaned up and quantified using a spectrophotometer. Sample purity was between 1.96 and 1.7 (A260/A280). Fragmented samples were then stored at –200C until hybridizations.
|
| |
|
| Hybridization protocol |
Hybridizations to the Affymetrix Drosophila 2.0 GeneChip microarray took place in the Microarray Core Facility at UC Davis.
|
| Scan protocol |
Hybridizations to the Affymetrix Drosophila 2.0 GeneChip microarray took place in the Microarray Core Facility at UC Davis.
|
| Description |
Sample 1 from this line
|
| Data processing |
Standard Affymetrix data processing
|
| |
|
| Submission date |
Aug 28, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Matthew W. Hahn |
| E-mail(s) |
mwh@indiana.edu
|
| Organization name |
Indiana University
|
| Department |
Department of Biology
|
| Street address |
1001 E. 3rd St.
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE8892 |
Abundant genetic variation in transcript level during early Drosophila development |
|
| Relations |
| Reanalyzed by |
GSE119084 |
