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| Status |
Public on Jun 02, 2009 |
| Title |
L1 stage larvae (Celniker/RNA:288) extraction3_array1 |
| Sample type |
RNA |
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| Source name |
L1 stage larvae (Celniker/RNA:288) extraction3_array1 channel_1
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Y cn bw sp
developmental stage: L1 stage larvae
genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1]
sex: Unknown
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| 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 ¼ 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.
All Drosophila larval collections were made from the sequenced D. melanogaster isogenic strain containing the mutations; yellow (y1), cinnabar (cn1), brown (bw1), and speck (sp1) [1]. First, second and early third instar larvae were staged and collected by this method. Synchronized embryos were collected from population cages that contained adult flies that were less than one week old and were maintained at 24 C. A two hour pre-lay was done prior to the start of timed collections each day. Embryos were collected for two hours on 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 (Baker?s Dry Yeast) and placed horizontally on a short 1cm raised Plexiglas bar in the bottom of each cage to avoid crushing flies. Embryos were aged 18 hours on the collection plates, washed from the plates with water, passed through an 850 micron screen to remove adults and collected on a 75 micron screen to remove yeast paste. Embryos were gently transferred onto a new hard egg-lay plate in rows and the rows surrounded by stripes of fresh yeast paste to tempt the larvae to migrate away from the unhatched embryos. The strips of unhatched embryos were cut out of the plate after a few hours and discarded. Feeding larvae in the yeast paste were aged appropriately and harvested by the screening procedure above. Larvae collected 42-44 hr after egg-lay were designated L1; larvae collected 66-70 hr after egg-lay were designated L2; larvae collected 83-85 hours after egg-lay were designated L3+12 hr. Collected larvae were washed briefly with de-ionized water in a buchner funnel, split into two samples and immediately frozen with dry ice. The staged animals were stored at minus 80oC for larval RNA production.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction: TRIzol procedure for Insects followed by DNase and RNeasyNote: This protocol is used to prepare RNA that will be used for microarray hybridization or RT-PCR.Materials TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml). DNase/RNase-free water: Invitrogen cat. no. 10977. 75% ethanol, prepared with DNase/RNase-free water. RNase-free DNase: Qiagen cat. no. 79254. RNeasy® spin columns: RNeasy midi kit, Qiagen cat. no. 75144. TenBroeck homogenizer: Bellco biotechnology cat no. 1982-10002.Procedure1. Homogenize insect samples in TRIzol reagent using a TenBroeck tissue homogenizer. The sample volume should not exceed 10% of the volume of TRIzol reagent used for homogenization.2. Incubate at room temperature for 5 minutes. 3. Aliquot samples into 1.5 ml microcentifuge tubes.4. Add 0.267 ml chloroform per ml of TRIzol used.5. Shake tubes vigorously for 15 seconds.6. Incubate at room temperature for 2 minutes.7. Centrifuge for 15 minutes at 4degC at 12,000 x g.8. Transfer the top (aqueous) phases to clean tubes.9. Precipitate the RNA from the aqueous phase by adding 0.67 ml of isopropanol per ml of TRIzol used in step 5.10. Invert tubes once to mix gently.11. Incubate samples at room temperature for 10 minutes.12. Centrifuge for 10 minutes at 4degC at 12,000 x g.13. Remove the supernatant.14. Wash RNA pellet once with 75% ethanol, using 0.7 ml per microcentrifuge tube.15. Vortex briefly.16. Centrifuge at 7,500 x g for 5 minutes at 4degC.17. Let the pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely.18. Dissolve in RNase-free water. 19. Incubate at 37degC overnight to dissolve the RNA. Determine concentration using a Nanodrop® ND-1000 Spectrophotometer. RNA may be stored at -80degC at this point if desired.20. Purify up to 1 mg of RNA on an RNeasy spin column, with DNase treatment, precisely as described in Qiagen's RNeasy Midi/Maxi Handbook, supplied with the RNeasy kit. Do the optional second wash on the column. Elute twice using DNase/RNase-free water.21. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. If necessary, the RNA can be concentrated on a SpeedVac. Store at -80degC.
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.
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| Label |
Biotin
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| 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.
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| Scan protocol |
Scanned at 0.7 microns/pixel.
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| Description |
n/a
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| Data processing |
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 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 genome version is r5
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| Submission date |
May 28, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
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| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
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| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL6629 |
| Series (1) |
| GSE16292 |
Dm_y[1]cn[1] bw[1] sp[1]_larval_L1_TotalRNA_p200_284-286-288_38bp |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM409540_Dro2_AS_Dm_L1_larvae_RW_C01_B3_T1.CEL.gz |
43.0 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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