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| Status |
Public on Mar 13, 2014 |
| Title |
Dmel_Canton-S_Male_Biorep2_Techrep1 |
| Sample type |
SRA |
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| Source name |
Dmel_Canton-S_Male_Biorep2_Techrep1
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w[1118], "Canton-S" "B"
developmental stage: Adult
gender: Male
tissue: Heads
genotype/variation: WT
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| Treatment protocol |
The Doa and fne mutant lines were backcrossed through 10 generations to the "Canton-S" line, in order to isogenize their genetic backgrounds. The mutant allele was selected at each generation based on phenotype (w+ in the case of fne; for Doa, whiteapricot was co-selected during backcrossing in order to allow selection of the Doa mutation at each generation). A TM6B balancer chromosome stock was also isogenized in the same way. This was crossed after the ten generations to the three Doa alleles (HD, DEM and EMS2) to re-create stable balanced stocks.
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| Growth protocol |
Drosophila were raised at 25o C under constant lighting (internal and room lighting) to ablate their circadian rhythm. Cultures were raised at low density during a minimum of 3 generations to minimize competition among larvae during development. Specifically, 4-5 males and 4-5 females were placed on fresh vials of standard corn-meal/yeast/agar medium and kept for two days before being changed to new vials. Adults for RNA isolation were collected once every 24 hours as soon as the first emergents were detected and placed on new vials with 50-100 flies/vial. Animals were aged for 7-8 days. 7-8 day old flies were flash frozen in dry-ice and stored at -80o C. Once significant quantities were available, frozen flies were sex-sorted on aluminum blocks kept on dry ice, and heads hand-dissected, collected and stored at -80 until homogenization for RNA preparation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Solexa library preparation was as follows: We fragmented mRNA using alkaline hydrolysis. First, 9 µl of 100 ng of mRNA, 1 ng of ERCC spike-in controls (Jiang et al., 2011) 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. Paired-end 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 PE primer 1.1, 1 µl PE 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 HiSeq 2000 |
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| Description |
WT_M
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| Data processing |
Basecalls performed using Illumina pipeline version 1.6.47.1 for runs on Illumina GAIIx, or CASAVA version 1.7 for runs on Illumina HiSeq 2000 instrument.
Reads that passed Illumina pipeline quality filtering criteria (those output to s_x_x_sequence.txt fastq files) were mapped to a reference genome.
Reads that passed Illumina pipeline quality filtering criteria (those output to s_x_x_sequence.txt fastq files) were mapped to a reference genome
Mapping was then performed using Tophat v1.4.1 (Trapnell et al., 2010), with Bowtie v0.12.7 (Langmead et al., 2010), and samtools 0.1.12a (Li et al., 2009), and parameters “-g 1 –solexa1.3-quals, -i 42.” A reference annotation (ENSEMBL release 62) was supplied in gtf format with the -G option.
The reference sequence for mapping was the dm3 assembly from the UCSC Genome Browser which corresponds to the April 2006 Drosophila melanogaster draft assembly from the Berkeley Drosophila Genome Project (Release 5), with sequence identified as “chrUextra” removed, and with sequence for ERCC exogenous controls (Jiang et al., 2011) added. See GSE20555 for control reference sequences.
BAM files were used to generate base level coverage in bedgraph format using Bedtools (Quinlan et al.), with the genomeCoverageBed command and the -bg option.
Genome_build: dm3
Supplementary_files_format_and_content: BEDGRAPH contain base-level coverage
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| Submission date |
May 07, 2012 |
| 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 |
GPL13304 |
| Series (1) |
| GSE37811 |
mRNA-Seq of head tissue from Doa and fne mutants, Drosophila melanogaster. |
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| Relations |
| Reanalyzed by |
GSM3277771 |
| SRA |
SRX146437 |
| BioSample |
SAMN00991393 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM928384_R57L5_WT_M.bedgraph.gz |
93.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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