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| Status |
Public on Jan 31, 2016 |
| Title |
Df(2L)ED629/+_male_rep2 |
| Sample type |
SRA |
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| Source name |
Whole animal
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Whole animal
gender: male
genotype: w[1118]; Df(2L)ED629, P{w[+mW.Scer\FRT.hs3]=3'.RS5+3.3'}ED629/+
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| Growth protocol |
The Drosophila deletion stocks were generated by DrosDel (http://www.drosdel.org.uk) as described in (Ryder et al., 2007). Flies were maintained at 25oC on standard yeast/cornmeal medium (Fly Facility, University of Cambridge, UK). We crossed males from DrosDel lines to w1118 virgin females to generate hemizygotes without balancer chromosomes. The w1118 line we used is the parental line for the DroDel project, so all flies are isogenic other than for the deleted region they carry. We prepared duplicates of 15-25 sexed Df/+ progeny (mean 21.65, standard deviation 7.4) 5 days post eclosion for RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Flies were homogenized in 500 μl of RNAlater solution (Life Technologies, Grand Island, NY, USA) for 90 seconds in 96 deep well plates with 3 mm tungsten carbide beads (Qiagen, West Sussex, UK) using a Qiagen Tissue Lyser II (Qiagen, West Sussex, UK). Extraction of total RNA was performed using the RNeasy 96 kit (Qiagen, Valencia, CA, USA) based on the manufacturer's guide (Protocol for Isolation of Total RNA from animal cells using QIAvac96 vacuum manifold, Cat#19504). We made a minor modification to dilute the RNAlater solution, so instead of mixing 150 μl of RLT buffer (Qiagen, Cat#79216) and 150 μl of 70% ethanol, we mixed 150 μl of RLT buffer, 120 μl of 100% ethanol, and 30 μl of lysate in RNAlater and applied this to the extraction columns.
Libraries were prepared using Illumina's (San Diego, CA, USA) TruSeq RNA sample preparation kit v2 - set A and B (Cat # RS-122-200). RNA amount was quantified using RiboGreen kits (Life Technology, Grand Island, NY, USA). 100 ng of RNA was used as input to follow the 1/2 scaled protocol from the original manufacturer's High Sample Protocol (Illumina, Part# 15026495 Rev. C). We added 10 pg (353 samples) or 500 pg (43 samples) of external RNAs as spike-in during the 8 minute RNA fragmentation step. The constructed libraries were sequenced with a HiSeq 2000 (Illumina, San Diego, CA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
95BM
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| Data processing |
Basecalls performed using CASAVA version 1.8.2.
RNA-Seq reads were aligned to the Drosophila genome assembly (Berkley Drosophila Genome Project Release 5, obtained from FlyBase. http://www.flybase.org) using TopHat 2.0.10 (Trapnell et al., 2009. Bioinformatics) with -g 1 and -G parameters. As a gene model for the -G parameter, we used an annotation file from FlyBase (5.57) where we removed genes from uncertain physical locations (e.g., chrU and chrUextra).
From the alignment result, abundance of transcripts were measured as read counts using HTseq (Anders et al., 2014. Bioinformatics) with default options (-m union -t exon).
From the alignment result, Fragments per Kilobase per Million mapped reads (FPKM) values were calculated using Cufflinks (Trapnell et al., 2010. Nature Biotechnology). We used -G, -b, and -u parameters in running Cufflinks.
Genome_build: Release 5
Supplementary_files_format_and_content: Tab-delimited text files that are generated from HTseq and Cufflink. Each contains read counts and FPKM values, respectively, at gene level.
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| Submission date |
Sep 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
[email protected]
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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) |
| GSE61509 |
Expression profiling pooled Drosophila melanogaster heterozygous for deletions on Chromosome 2L |
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| Relations |
| Reanalyzed by |
GSM3287447 |
| BioSample |
SAMN03072277 |
| SRA |
SRX703219 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1507176_fpkm_95BM.txt.gz |
467.0 Kb |
(ftp)(http) |
TXT |
| GSM1507176_htseq_95BM.txt.gz |
61.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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