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| Status |
Public on Jun 02, 2009 |
| Title |
embryos,12-14 hr after egg laying (Celniker/RNA:422) extraction2_array1 |
| Sample type |
RNA |
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| Source name |
embryos,12-14 hr after egg laying (Celniker/RNA:422) extraction2_array1 channel_1
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Y cn bw sp
developmental stage: Embryo 12-14h
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.
''This protocol was used for staging and collection of Drosophila melanogaster embryos by Dave Miller''''(Kaufman Lab, IU Bloomington) for transcriptome analysis by the Celniker group.''All Drosophila tissue collections were made from the sequenced ''D. melanogaster'' isogenic strain containing the mutations; ''yellow'' (y1), ''cinnabar'' (cn1), ''brown'' (bw1), and ''speck'' (sp1) [1]. Synchronized embryos were collected from population cages that were less than one week old and maintained at 24 C. At least one ? two hour pre-lay was made 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 (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.Staged embryos were passed through an 850 micron screen and collected on a 75 micron screen to remove adults and yeast paste. Embryos were then dechorionated by treatment with 50% bleach (3% sodium hypochlorite), 0.2% sodium chloride, and 0.02% Triton-X-100 for five minutes. Embryos were washed twice with 0.2% NaCl, 0.02% Triton buffer and split into two samples. Most of the sample (approximately 95%) was rinsed with de-ionized water in a buchner funnel under mild vacuum, dried briefly, immediately frozen on dry ice and stored at -80 C for RNA preparations. The small aliquot was transferred to a clean tube and fixed for staging. Fixation was accomplished in 0.1 M Pipes (pH 6.9), 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde, 0.1% glutaraldehyde (percentages are relative to aqueous volumes), and 50% heptane, as describe previously [2]. Samples were shaken for five minutes in the fixative, centrifuged briefly and the aqueous fraction was removed. An equal volume of methanol containing 2 mM EGTA was added and the sample was shaken for five additional minutes. Tissue was washed twice in methanol with 2 mM EGTA and saved at -80 C for the characterization of developmental stages. Fixed embryos were used to determine the embryonic stages present in each biological sample X (BS#X). Each biological sample was washed in 0.2% sodium chloride, and 0.02% Triton-X-100 for five minutes and placed in a P0042G Sedgewick Rafter Grid slide. The developmental stages present in each sample were counted under a dissecting scope. The criteria used for stage identifications are describe in detail at FlyBase [3] and Campos Ortega, J. A. and Hartenstein, V (1997)[4]. Embryonic stages were not assessed by morphology associated with head involution due to the observed defects in this process associated with the isogenic strain. Data were entered into a Microsoft Exel worksheet for graphing. Biological samples that required pooled collections for adequate RNA yields were staged independently and normalized according to the fraction of the final mass of tissue in the pooled biological sample
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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.
Poly (A)+ RNA isolation: Qiagen Oligotex mRNA Spin-Column procedureThis protocol is used to prepare poly (A)+ RNA from total RNA. It utilizes the Oligotex mRNA Midi kit (Qiagen Cat# 70042), and the procedure is unmodified from Qiagen Oligotex Handbook Second Edition, May 2002.Materials? Oligotex mRNA Midi kit (Qiagen Cat# 70042)? Oligotex Suspension: 10% (w/v) suspension Oligotex particles in: 10 mM Tris?Cl, pH 7.5; 500 mM NaCl; 1 mM EDTA; 0.1% SDS; 0.1% NaN3 (sodium azide)? Wash Buffer OW2: 10 mM Tris?Cl, pH 7.5; 150 mM NaCl; 1 mM EDTA? Elution Buffer OEB: 5 mM Tris?Cl, pH 7.5? Buffer OBB: 20 mM Tris?Cl, pH 7.5; 1 M NaCl; 2 mM EDTA; 0.2% SDSProcedure1. Determine the concentration of the starting total RNA solution. Add 250 ? 500 microgram total RNA (maximum volume 500 microliters) to a 1.5 ml microcentifuge tube and adjust the volume to 500 microliters with RNase-free water.2. Add 500 microliters buffer OBB and mix.3. Add 30 microliters Oligotex suspension (ensure that the resin is in suspension), and mix.4. Incubate the sample for 3 minutes at 70degC in a heating block.5. Remove the sample from heating block and place at 20 to 30degC for 10 minutes.6. Centrifuge for 2 minutes at 15,000 g to pellet the Oligotex:mRNA, and carefully remove the supernatant.7. Resuspend the Oligotex:mRNA pellet in 400 microliters of Buffer OW2 by pipetting up and down. Transfer the suspension onto a small spin column placed in a 1.5 ml microcentrifuge tube.8. Centrifuge for 1 minute at 15,000 g.9. Transfer the spin column to a new RNase-free 1.5 ml microcentrifuge tube, and apply 400 microliters of Buffer OW2 to the column.10. Centrifuge for 1 minute at 15,000 g. 11. Transfer the spin column to a new RNase-free 1.5 ml microcentrifuge tube. Add 50-80 microliters pre-heated (70degC) Buffer OEB to the column and resuspend the resin by pipetting up and down 3 or 4 times.12. Centrifuge for 1 minute at 15,000 g.13. Add another 40-50 microliters pre-heated (70degC) Buffer OEB onto the column resuspend the resin by pipetting up and down 3 or 4 times.14. Centrifuge for 1 min at 15,000 g. 15. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. 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) |
| GSE16307 |
Dm_y[1]cn[1] bw[1] sp[1]_embryo_12-14h_poly-Ap_p200_421-422-423_38bp |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM409584_Dro2_AS_Dm_emb_14h_RWP+_C01_B2_T1.CEL.gz |
45.9 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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