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| Status |
Public on Aug 02, 2012 |
| Title |
Tdc2 rep2 |
| Sample type |
SRA |
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| Source name |
Octopaminergic cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Head
cell type: Tdc2-GAL4 cells
age: Adult
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Adult flies were anesthetized by CO2 and flash frozen in liquid N2. Heads were separated from abdominal-thoracic segments, wings and legs by vigorous vortexing followed by separation over dry ice cooled sieves. 600 - 10,000 frozen heads were added to 50mls of 10mM b-glycerophosphate pH7, 2mM MgCl2, 5mM sodium butyrate, 1X complete protease inhibitor cocktail (Roche: 11873580001) and the suspension was passed over a Yamato continuous flow homogenizer, set at 90-100 rpm, five to seven times. The homogenate was filtered over Miracloth (EMD Biosciences: 475855) and bought to 0.7mM b-mercaptoethanol and 0.5% NP-40. After six tractions in a 40ml Dounce homogenizer (pestle B), 600uls of antibody-adsorbed beads were added to 50mls of lysate. The binding reaction was run under continuous agitation at 4oC for 30 minutes. Beads were then collected on a magnet and washed three to four times in 50mls 10mM β-glycerophosphate pH7, 250mM sucrose, 2mM MgCl2, 25mM KCl, 5mM sodium butyrate. Bead bound nuclei in 20mls wash buffer were then passed over a 20um nylon mesh, returned to the magnet stand and resuspended in 1ml of 10mM β-glycerophosphate pH7, 250mM sucrose, 2mM MgCl2, 25mM KCl, 5mM sodium butyrate. Bead bound nuclei were collected on a magnet stand, then resuspended in 400μls of 100mM Tris pH7, 4M guanidinium thiocyanate. After 30 minutes of agitation at 4oC the supernatant containing nuclear RNA was removed from the beads and extracted with an equal volume of phenol:CHCl3. After the addition of 0.1 volume 3M sodium acetate pH5, the sample was extracted with acid phenol. The Agencourt RNA-Advantage kit (Beckman Coulter: 47942) was then used to purify RNA directly from the aqueous layer of the last extraction. Nuclear RNA was then converted to cDNA using a Nugen Ovation RNA-seq v2 kit (Nugen: 7102). Amplified cDNA was then sheared in a Corvaris s2 instrument, end-repaired, linker-adapted and sequenced on an Illumina HiSeq 2000 to 50bp read length. RNA was isolated from whole dissected brains in 100mM Tris pH7, 4M guanidinium thiocyanate.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Tdc2-GAL4 (Cole et al., 2005) driving unc84-GFP, primarily in octopaminergic neurons
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| Data processing |
Base calling performed by Illumina CASAVA 1.8
Five base pairs were trimmed from 5'-end of all reads to remove possible adaptor contamination, using FASTX toolkit v0.0.13 (fastx_trimmer -f 6 -z -Q33)
Reads were aligned to dm3/ERCC92 combined index using TOPHAT v1.4.0 (--num-threads 8 --GTF flybase_r5.41_ERCC92.gtf --no-novel-juncs --min-intron-length 30 --max-intron-length 200000)
Expression levels (FPKM) and differential expression were estimated using CUFFDIFF v 1.3.0 (cuffdiff --num-threads 8 --multi-read-correct --frag-bias-correct dm3_ercc92.fa -M $MASK_GTF)
Coverage over genomic positions counted with BEDTOOLS v2.15 (genomeCoverageBed -split -bg)
Coverage scaled to 10M total alignments. (custom Perl script)
Scaled coverage was converted to bigWig (wigToBigWig from ucsc)
Genome_build: dm3
Supplementary_files_format_and_content: bigWig tracks for visualization; supplemental files are tab-delimited files containining CUFFDIFF estimated expression levels and differential expression from pairwise comparisons for (1) annotated isoforms and (2) genes.
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| Submission date |
Apr 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Fred P Davis |
| E-mail(s) |
[email protected]
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| Phone |
571-209-4000-3437
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| Organization name |
HHMI Janelia Farm Research Campus
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| Street address |
19700 Helix Dr
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| City |
Ashburn |
| State/province |
VA |
| ZIP/Postal code |
20147 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE37027 |
Cell type-specific gene expression profiling of Drosophila neurons [RNA-Seq] |
| GSE37033 |
Cell type-specific gene expression and chromatin profiling of Drosophila neurons |
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| Relations |
| Reanalyzed by |
GSM3277327 |
| SRA |
SRX135207 |
| BioSample |
SAMN00849449 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM909109_adult_Tdc2_brain_120202_rna_scaled10M-tophat.bw |
173.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data are available in SRA |
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