|
| Status |
Public on Mar 31, 2015 |
| Title |
whole embryo, actD treat, 1 hour pulse |
| Sample type |
RNA |
| |
|
| Source name |
whole embryo, actD treat, 1 hour pulse RNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: whole embryo
genotype: wildtype
developmental stage: embryo
embryonic stage: stage 12 - 15
strain: UAS-T.g.UPRT (no GAL4)
|
| Treatment protocol |
Embryos were permeabilized and pulse-labeled in tagging media [D22 insect media, 5% FBS, and either 1mM 4-thiouridine (whole embryo tagging) or 1mM 4-thiouracil (neural-specific tagging)] for 1 hour at 30°C. Pulse-labeled embryos were then transferred for 1 or 3 hours to chase media containing uridine [D22 insect media, 5% FBS, 10mM uridine] at 30°C. Embryos were homogenized for 30 seconds and frozen in Trizol (Invitrogen) at -80°C. Multiple samples for each time-point were pooled for RNA extraction and TU-RNA purification, as previously described
|
| Growth protocol |
embryos were collected on standard apple juice, agar plates and reared at 18C or 25C to reach stage 12 - 15
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using trizol purification protocols. TU-RNA was purified as previously described.
|
| Label |
Cy3
|
| Label protocol |
Purified TU-RNA plus a spike-in control (One-Color RNA Spike-In Kit, Agilent) was used to generate Cy3 labeled cRNA (Low Input Quick Amp Labeling Kit, Agilent) and hybridized to Agilent 4x44K Drosophila gene expression microarrays.
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| |
|
| Hybridization protocol |
1.65 micrograms of Cy3 labeled cRNA was combined with the recommended dilution of spike-in control and hybridized following manufacturer's instructions
|
| Scan protocol |
Slides were scanned immediately after washing on the GenePix 4000B scanner, with pixel size 10, scan power 100%, and 532nm PMTGain set between 500 and 700.
|
| Description |
TU-RNA present after 1 hour pulse
|
| Data processing |
Spot alignments and data measurements were made using GenePix Pro 5.1 software. Post-processing of GenePix Pro data was performed in R.
Signal intensities within a pulse-chase series were normalized using the spike-in control. Threshold F532 intensity (mean - background) for a spot to be considered present (signal above background) was determined for each pulse-chase series by averaging the signals for maternal mRNAs that should not be present in the TU-RNA samples at any timepoint. Spots with signals below this average value were removed from analysis.
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| |
|
| Submission date |
Mar 30, 2015 |
| Last update date |
Mar 31, 2015 |
| Contact name |
Michael Cleary |
| E-mail(s) |
mcleary4@ucmerced.edu
|
| Organization name |
University of California, Merced
|
| Department |
School of Natural Sciences
|
| Street address |
5200 N. Lake Rd.
|
| City |
Merced |
| State/province |
CA |
| ZIP/Postal code |
95343 |
| Country |
USA |
| |
|
| Platform ID |
GPL6385 |
| Series (1) |
| GSE67435 |
Genomewide measurements of whole embryo mRNA decay in Drosophila |
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