|
| Status |
Public on Dec 03, 2011 |
| Title |
lola_isoform_K_(ORC4)_rep7 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ctrl embryos 10-12 hours after egg laying
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w1118; sca-GAL4/UAS-tau-eGFP (control)
developmental stage: embryonic, 10-12 hours after egg laying
|
| Treatment protocol |
Collected embryos were dechorionated with 50% bleach, rinsed once with 0.1 M Na phosphate pH 7.2; 0.3% Triton X-100, and rinsed twice with sterile water.
|
| Growth protocol |
Embryos were collected for 2 hours at 25C on grape juice agar plates and incubated an additional 6 hours at 25C in a moist chamber. Embryos of the desired genotype were then hand-sorted with a fluorescent dissecting microscope based on positive GFP expression in the CNS. Sorted embryos were returned to 25C and allowed to develop until 10.0 hours after the end of egg collection. lola mutant and control embryos were collected concurrently.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental and control total RNA samples were extracted with Trizol (Invitrogen) per manufacturer’s instructions. Final RNA pellet was rinsed twice with 75% ethanol, air-dried 10 minutes, resuspended in 20μl RNase-free water at 60o and stored at -70o. Each RNA sample for microarrays was 20-30 μg of total RNA, derived from ~300 embryos.
|
| Label |
Cy3
|
| Label protocol |
First-strand cDNA synthesis and labeling were performed using the Fred Hutchinson Cancer Research Center Genomics Resources standard protocol. In brief, each total RNA sample (20-30 μg) was reverse transcribed using SuperScript II (Invitrogen) with aa-dUTP/dNTPs incorporation. The resulting cDNAs were subsequently coupled to either Cy3 or Cy5 fluorophores (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
|
| |
|
| Channel 2 |
| Source name |
lola C4-/- embryos, 10-12 hours after egg laying
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w1118; lolaORC4 sca-GAL4/lolaORC4 UAS-tau-eGFP
developmental stage: embryonic, 10-12 hours after egg laying
|
| Treatment protocol |
Collected embryos were dechorionated with 50% bleach, rinsed once with 0.1 M Na phosphate pH 7.2; 0.3% Triton X-100, and rinsed twice with sterile water.
|
| Growth protocol |
Embryos were collected for 2 hours at 25C on grape juice agar plates and incubated an additional 6 hours at 25C in a moist chamber. Embryos of the desired genotype were then hand-sorted with a fluorescent dissecting microscope based on positive GFP expression in the CNS. Sorted embryos were returned to 25C and allowed to develop until 10.0 hours after the end of egg collection. lola mutant and control embryos were collected concurrently.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Experimental and control total RNA samples were extracted with Trizol (Invitrogen) per manufacturer’s instructions. Final RNA pellet was rinsed twice with 75% ethanol, air-dried 10 minutes, resuspended in 20μl RNase-free water at 60o and stored at -70o. Each RNA sample for microarrays was 20-30 μg of total RNA, derived from ~300 embryos.
|
| Label |
Cy5
|
| Label protocol |
First-strand cDNA synthesis and labeling were performed using the Fred Hutchinson Cancer Research Center Genomics Resources standard protocol. In brief, each total RNA sample (20-30 μg) was reverse transcribed using SuperScript II (Invitrogen) with aa-dUTP/dNTPs incorporation. The resulting cDNAs were subsequently coupled to either Cy3 or Cy5 fluorophores (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed using the Fred Hutchinson Cancer Research Center Genomics Resources standard protocol. Briefly, experimental and control labeled cDNA targets were co-hybridized to microarrays for 16 hrs at 63°C and sequentially washed at room temperature in 1 x SSC and 0.03% SDS for 2 min, 1 x SSC for 2 min, 0.2 x SSC with agitation for 20 min, and 0.05 x SSC with agitation for 10 min. Arrays were immediately centrifuged until dry.
|
| Scan protocol |
Arrays were scanned using a GenePix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA). Image analysis was performed using GenePix Pro 6.0.
|
| Description |
ctrl_S8_vs_lolaC4_s8_n4038
|
| Data processing |
LOESS normalized, no background correction, scaled by median absolute deviation, all performed using limma package (Author, Gordon Smyth) within R statistical language.
|
| |
|
| Submission date |
Dec 02, 2011 |
| Last update date |
Dec 07, 2011 |
| Contact name |
Edward Giniger |
| E-mail(s) |
ginigere@ninds.nih.gov
|
| Organization name |
NIH
|
| Department |
NINDS
|
| Lab |
AGNCU
|
| Street address |
Bldg 35, Rm 1C-1002
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1908 |
| Series (2) |
| GSE34122 |
A genome-wide analysis reveals that the Drosophila transcription factor, Lola, promotes axon growth in part by suppressing expression of the actin nucleation factor, Spire (allele lola_ORC4) |
| GSE34123 |
A genome-wide analysis reveals that the Drosophila transcription factor, Lola, promotes axon growth in part by suppressing expression of the actin nucleation factor, Spire |
|
