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| Status |
Public on Apr 08, 2010 |
| Title |
Head_Dmel_Female (GA II) |
| Sample type |
SRA |
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| Source name |
head tissue
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: OregonR
tissue: head
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| Treatment protocol |
Frozen whole flies were placed on 10.8 x 10.8 cm ceramic plates frozen on dry ice. Forceps were used to dissect 400-500 heads/sex for subsequent RNA extraction.
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| Growth protocol |
Drosophila melanogaster (OregonR) were grown at 22°C, 60% relative humidity in standard plastic vials containing cornmeal media. All media was purchased from the Tucson Stock Center, Tucson, Arizona (recipes: http://flyfood.arl.arizona.edu/recipes.php3). There were no lights inside of incubators and room lighting (standard fluorescent bulbs) was on at all times. Newly eclosed flies of both females and males were housed together in vials at normal density (60-100 flies) and aged for 7 days. On day 7, flies were anesthetized with CO2 and then females and males were sorted and placed into separate vials. Flies in same sex vials were aged an additional 24 hours then flash frozen on dry ice in 50 ml Falcon tubes and stored at -80°C until dissection.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Fly heads were immersed in 250 µl of TRIzol and thoroughly homogenized using a 1.5 ml RNAase free pestle (Kimble-Chase, Vineland, New Jersey) fitted to a Pellet Pestle Motor (Sigma-Aldrich, St. Louis, Missouri). After initial homogenization, 550 µl of TRIzol was added and the solution was further homogenized by hand. Chloroform (160 µl) was added to the homogenate and then the sample was centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous layer was removed, precipitated with isopropyl alcohol and glycogen (8 µg), then centrifuged for 10 minutes at 4°C to pellet the RNA. The supernatant was removed and the RNA pellet was washed with fresh 75% ethanol. The pellet was dried briefly and then dissolved in 50 µl DEPC treated H2O. mRNA was enriched from total RNA by poly A+ selection using a QIAGEN Oligotex mRNA kit (Valencia, California) following manufacture instructions. mRNA was eluted from small spin columns twice with hot 30 µl buffer OEB. mRNA quality and quantity was determined by Bioanalyzer (Agilent, Santa Clara, California) and 260/280 ratio from Nanodrop ND-1000 UV spectrophotometer.
We fragmented mRNA using alkaline hydrolysis. First, 9 µl of 100 ng of mRNA and 1 µl of 10X fragmentation buffer (Ambion, Austin, Texas.) was incubated at 70°C for 5 minutes to fragment mRNA. One microliter of Stop Buffer (Ambion, Austin, Texas) was added and samples were placed on ice. Fragments were precipitated with 1 µl 3 M NaOAC (pH 5.2), 2 µl glycogen (5 µg/µl , Ambion, Austin, Texas), and 30 µl 100% EtOH and then incubated for 30 minutes at -80 °C. Fragmented RNA was pelleted at 14000 rpm with a microcentrifuge at 4°C. The pellet was washed with 70% EtOH, air dried and resuspended with 10.5 µl RNase free H2O. Fragmented mRNA was reverse transcribed to create cDNA. One microliter of random hexamer primers (3µg/µl, Invitrogen, Carlsbad, California) was placed with 10.5 µl of fragmented mRNA, then samples were incubated at 65°C for 5 minutes and placed on ice. Four microliters 5X first strand buffer, 2 µl 100 mM DTT, 1 µl dNTP, and 0.5 µl RNaseOUT were added to the mRNA and samples were incubated at 25°C for 2 minutes. One microliter of SuperScript II (200 U/µl, Invitrogen, Carlsbad, California) was added and samples were incubated under the following conditions: 25°C-10 minutes, 42°C-50 minutes, 70°C-15 minutes. Samples were then placed on ice. Second strand cDNA was synthesized by adding 61µl H2O to the first strand mix, 10 µl 10X second strand buffer (500 mM Tris-HCl pH 7.8, 50 mM MgCl2, 10 mM DTT), and 3 µl dNTP (10 mM), mixed, incubated on ice for 5 minutes, then 1 µl RNaseH (2U/µl, Invitrogen, Carlsbad, California) and 5 µl DNA Pol I (10 U/µl, Invitrogen, Carlsbad, California) were added. Samples were incubated at 16°C for 2.5 hours and DNA products purified using a QIAquick spin column and following manufacture directions and eluted with 30 µl. cDNA fragment libraries were prepared for sequencing. The ends of cDNA fragments were repaired using 30 µl of DNA, 45 µl H2O, 10 µl T4 DNA ligase buffer with 10 mM ATP, 4 µl dNTP mix (10 mM), 5 µl T4 DNA polymerase (3 U/µl), 1 µl Klenow DNA polymerase (5U/µl), and 5 µl T4 PNK(10 U/µl). Samples were incubated for 30 minutes at 20°C, purified with QIAquick PCR spin columns, and eluted with 32 µl of Buffer EB. A single “A” base was added to the ends of cDNA by using 5 µl Klenow buffer, 10 µl dATP (1 mM) and 3 µl Klenow 3’ to 5’ exonuclease (5 U/µl). Samples were incubated at 37°C for 30 minutes, purified with a QIAquick MiniElute column, and eluted with 19µl of EB solution. Adaptor oligonucleotdies were ligated to A overhang fragments as follows. Overhang fragments were combined with 25 µl DNA ligase buffer, 1 µl adaptor olio mix, and 5 µl DNA ligase (1U/µl). Samples were incubated at room temperature for 15 minutes, purified with a QIAquick MiniElute column, and eluted with 10 µl of buffer EB. cDNA templates were sized selected by gel purification then amplified by PCR using oligo-specific primers. Samples were loaded into a 2% agarose gel with 1X TAE buffer and run for 50 minutes with a 100 bp ladder. The gel was stained with ethidium bromide and bands were visualized under UV fluorescence. For each sample, a gel slice was cut at the 200 (+/- 25) bp region, purified with a QIAquick gel extraction kit, and eluted with 30 µl buffer EB. Purified cDNA was amplified in a 50 µl reaction using 25 µl Phusion Mix (Phusion polymerase, dNTPs, and buffer), 1 µl primer 1.1, 1 µl primer 2.1, and 23 µl template along with the following cycling conditions: initial denaturation 98°C-30 sec, cycle denaturation 98°C-10 sec, anneal 65°C-30 sec, and extend 72°C -30 sec with 15X cycles and a final extension of 72°C for 5 minutes followed by a hold at 4°C. The amplified product was purified using a QIAquick column and eluted with 30 µl buffer EB.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
n/a
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| Data processing |
Reads that passed default parameters of the Illumina quality filter were mapped with Bowtie 0.11.3. The reference sequence used to built an index for mapping was the dm3 from the UCSC Genome Browser which corresponds to the April 2006 Drosophila melanogaster draft assembly from the Berkeley Drosophila Genome Project (Release 5). The reference index was created using the bowtie-build function with default parameters. Reads were mapped using -v 2 -m 1 in Bowtie v. 0.11.3.
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| Submission date |
Feb 16, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
briano@nih.gov
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| Phone |
301-204-9463
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| Organization name |
NIDDK, NIH
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| Department |
LBG
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| Lab |
Developmental Genomics
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| Street address |
50 South Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL9061 |
| Series (2) |
| GSE20348 |
mRNA-Seq of head tissue from Drosophila melanogaster |
| GSE44612 |
Comparative Validation of the D. melanogaster Encyclopedia of DNA Elements Transcript Models |
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| Relations |
| Reanalyzed by |
GSM3274597 |
| SRA |
SRX018864 |
| BioSample |
SAMN00010988 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM509821_Dmel_R23s_6_bowtie_75.txt.gz |
108.6 Mb |
(ftp)(http) |
TXT |
| GSM509821_Dmel_R33s_1_bowtie_75.txt.gz |
193.6 Mb |
(ftp)(http) |
TXT |
| GSM509821_Dmel_R33s_2_bowtie_75.txt.gz |
233.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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