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| Status |
Public on Mar 23, 2011 |
| Title |
Whole_Dpse_111.94_Male_Rep1 |
| Sample type |
SRA |
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| Source name |
Whole tissue
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| Organism |
Drosophila pseudoobscura |
| Characteristics |
strain: Drosophila Species Stock Center (DSSC) 14011-0121.94. See Drosophila 12 Genomes Consortium. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450:203-218 for more details about strain origin.
tissue: Whole flies
developmental stage: Adult
sex: Male
age: 5-7 days post eclosion
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| Treatment protocol |
Flies were raised in incubators with no photoperiod at 22 C and 60% humidity. See GSE6640 for details.
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| Growth protocol |
Adult flies were growth 5-7 days post eclosion. See GSE6640 for details.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
See GSE6640 for RNA extraction methods. We fragmented mRNA using alkaline hydrolysis. First, 9 µl of 100 ng of mRNA, 1 ng of ERCC spike-in controls, 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 300 (+/- 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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| Data processing |
Reads were trimmed to 75 bp due to low quality on the 3' end of the 101 bp reads. Trimmed 75 bp reads were used for subsequent analyses. Trimmed reads that passed the Illumina filter were mapped with Tophat 1.1.4. The species-specific reference sequence plus ERCC spike-in control sequences (see GEO entry GSE20555 for spike-in control sequences) was used to build a reference index for mapping. The reference index was created using the bowtie-build function with default parameters and reads were mapped to this reference using the Tophat parameters -g 1 -F 0 -r 150 -solexa1.3-quals.
supplementary_file_build: dp4+ERCC Spike-In Controls
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| Submission date |
Mar 21, 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 |
GPL9958 |
| Series (2) |
| GSE28078 |
mRNA-Seq of whole flies from Drosophila |
| GSE44612 |
Comparative Validation of the D. melanogaster Encyclopedia of DNA Elements Transcript Models |
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| Relations |
| SRA |
SRX054482 |
| BioSample |
SAMN00254133 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM694281_R54s4_Dpse_111.94_M_R1.bam |
1.1 Gb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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