|
| Status |
Public on Aug 05, 2010 |
| Title |
Celniker/RNA:516 extraction2_array1 |
| Sample type |
RNA |
| |
|
| Source name |
Celniker/RNA:516 extraction2_array1 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon-R
tissue: Female heads
developmental stage: Adult female, eclosion + 1 day
genotype: wild type
sex: Female
|
| Growth protocol |
Fly population cages contained the sequenced D. melanogaster isogenic strain with the mutations; yellow (y1), cinnabar (cn1), brown (bw1), and speck (sp1) [1]. Two homemade Plexiglas cages approximately 25cm x 25cm x 25cm, were used for staged embryo collections. These population cages were constructed from ΒΌ inch solid Plexiglas except for the open front panel. The fronts of the cages were covered with sleeves constructed of muslin cloth that were attached to the Plexiglas sides using Velcro strips. Each muslin sleeve was then twisted and held closed with a hose clamp. Flies were fed with three hard egg lay collection plates made in 150 X 15 mm Petri dishes containing a substrate of 3.3% agar, 13% unsulfured molasses, and 0.15% Tegasept. The hard egg lay plates were completely covered with a thin layer of moist yeast paste (Fleischmann?s Baker?s Dry Yeast) and placed horizontally on a short 1cm raised Plexiglas bar in the bottom of each cage to avoid crushing flies. The two population cages were maintained at 24? C in a controlled environment on a 24-hour light cycle (14 hours light / 10 hours dark) using three 60W bulbs on a timer. Flies were introduced to the cages at least four days prior to collections and fertility counts. These animals were initially grown in bottles at the same controlled environment described above. Each population cage was established from four trays with 35 bottles per tray containing newly eclosed adult flies. Each 250ml (pint) bottle contained approximately 40ml standard Drosophila medium in use at Bloomington (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm). The isogenic Drosophila mutant strain was propagated in bottles (A) starting with approximately 40-50 adults per bottle on day zero. New bottles (B) were generated from these bottles (A) on day 14. Cages were established by clearing the ?A? bottles into an empty bottle (without food) and transferring the newly eclosed adult flies in the ?A? bottles into the two population cages on day 18. The next round of cages and bottles (C) were established from the ?B? bottles in the same manner. Two-hour embryo collections were made during the light cycles following at least one two-hour pre-lay.
Protocol for the separation of heads from adult flies (aged 1-20 days post-eclosion). Note that this protocol typically results in loss of antennal and maxillary organs but retention of mouthparts.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Protocol for isolating total RNA from dissected tissues of Drosophila.
First-strand cDNA synthesis was performed by using SuperScript II reverse transcriptase in the reaction volume of 105 ul for 15 ug of starting RNA material. The RNA was mixed with random hexamers (83.3 ng/g mRNA), heated to 70C for 10 min, and cooled to 15C after which 5x SuperScript II First Strand buffer, DTT (10 mM), and dNTPs (0.5 mM) were added. SuperScript II was added after a 20-min incubation (200 units/ug RNA) followed by a 20-min ramp to 42C and 60-min incubation at 420C. SuperScript II was inactivated at 75C for 15 min.The second-strand cDNA was synthesized by addition of 50 units of Escherichia coli DNA ligase, 200 units of E. coli DNA polymerase I, 10 units of E. coli RNase H, and 0.2 mM dNTPs to the first-strand synthesis reaction at 16C for 2 h.
|
| Label |
Biotin
|
| Label protocol |
Double-stranded cDNA was treated with RNase H (Epicentre Technologies) and RNases A/T1 (Ambion), extracted by using a QIAquick PCR purification kit (Qiagen), and subjected to further fragmentation to 50-100 bp by DNase I (1 unit/ul; Epicentre Technologies; size distribution of fragmented DNA was verified on a 2% agarose gel).The fragmented cDNA was then end-labeled with 2.5mM biotinylated DNA labeling reagent (Affymetrix) by using 100 units of terminal deoxynucleotidyltransferase (TdT; Roche Diagnostics) in 1x TdT buffer (Roche Diagnostics) and 5 mM CoCl2 (Roche Diagnostics) for 2 h at 37C.
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| |
|
| Hybridization protocol |
For array hybridization, 2ug of the labeled DNA material was hybridized per chip for 18 h at 45C in a 3 M tetramethyl ammonium chloride/1x MES-based solution containing 100?g/mL Herring Sperm DNA, 0.02% Triton and 30pM of biotinylated control oligo B2 (Affymetrix). All reagents were from Invitrogen, except where noted otherwise.
|
| Scan protocol |
Scanned at 0.7 microns/pixel.
|
| Data processing |
Data_Normalization_for_Expression_Arrays protocol. Three biological replicates were hybridized for this experiment. The data from replicate arrays were quantile-normalized (Bolstad et al., 2003) and all arrays were scaled to a median array intensity given (e.g. 361). Processed data are obtained using following parameters: median value is 361 Signal Graph generation protocol. The sliding window approach (bandwidth 50) has been used to estimate RNA abundance (signal which is listed in column #2). It was found by calculating the median of all pairwise average PM-MM values, where PM is a perfect match and MM is a mismatch. Processed data are obtained using following parameters: bandwidth is 50
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| |
|
| Submission date |
Jul 28, 2010 |
| Last update date |
Aug 05, 2010 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6629 |
| Series (1) |
| GSE23233 |
Dm_4dFVirginHead_TotalRNA_515_516_38bp |
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