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| Status |
Public on Apr 03, 2012 |
| Title |
Wholeflies_ED499_Female_R2_Illumina |
| Sample type |
SRA |
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| Source name |
Wholeflies, 5 day mated adults
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| Organism |
Drosophila melanogaster |
| Characteristics |
gender: Female
strain: Df(2L)ED499/+
tissue: whole flies
developmental stage: 5 day old mated adults
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| Treatment protocol |
We collected replicates for experiments by outcrossing the balancer chromosome with the original wild-type stock (w1118) as follows; 18-24 vials were setup with 15 less than 8 day old virgin w1118 females paired with 5 less than 8 day old males of a particular deficiency line per vial. Df/+ female and males were placed together in vials at less than 60 per vial and then aged for five days. On day five, flies were anesthetized with CO2, sorted into males and females, then placed into 1.5 ml Eppendorf tubes and flash frozen on dry ice. All flies aged together for 5 days constituted a replicate. Flash frozen flies were stored in -80°C before RNA extraction.
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| Growth protocol |
Flies were grown under constant light, temperature, and humidity (25°C; 60% relative humidity) on San Diego Stock Center cornmeal media (DSSC, 2008)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We obtained 8 clones and the preparation protocol found in Mortazavi et al. 2008. Nat. Methods 5:621-628 (kindly provided by the Wold lab) and prepared transcripts by in vitro transcription using the Ambion MEGAScript kit (Ambion, Austin, Texas). We checked for full length transcripts using MOP gels and measured RNA concentration using the Nanodrop ND-1000 UV spectrophotometer. We made cocktails of these 8 spike-in controls by performing dilutions to obtain a range of concentrations from 5 x 108 to 1 x 102 transcripts (Gene: Number of Transcripts: OBF-1:5x108; VATG:5x107; Lambda1-1:5x106; Apetala- 2:1x106; AGP-2:5x105; EPR-1:1x105; Lambda2-3:5x104; PDF:1x102). We fragmented mRNA using alkaline hydrolysis. First, 9 μl of 100 ng of mRNA along with 1 ng of eight spike-in controls from the Wold lab (Mortazvi et al. 2008) 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 61 μl 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. 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 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 - 300 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. Libraries were prepared for 36 bp single end Illumina sequencing following standard protocols for clustering and imaging.
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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 |
Same poly A mRNA prep used for microarray hybridization
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| Data processing |
Reads that passed the default parameters of the Illumina quality filter were mapped with Bowtie. We combined the sequences of the spike-in controls and 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) to create a reference index for mapping. The reference index was created using the bowtie-build function with default parameters. Reads were mapped using -v 2 - m 1 in Bowtie v. 0.12.7 and then unique mapping sequences that mapped to spike-in controls and Flybase gene models were counted using the union mode in HTSeq. We used the unique mapping reads to calculated the Reads Per Million Mapped Reads (RPKM) as the normalized metric of gene expression and for comparison to intensities from microarrays.
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| Submission date |
Aug 19, 2011 |
| 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) |
| GSE31407 |
Drosophila DrosDel deficiency and diploid control flies |
| GSE31549 |
RNA-Seq on Illumina platform of Drosophila DrosDel deficiency lines |
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| Relations |
| Reanalyzed by |
GSM3274822 |
| SRA |
SRX092564 |
| BioSample |
SAMN00710509 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM783087_R35s_2_bowtiemap_v2m1_36.sam.gz |
335.2 Mb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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