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| Status |
Public on Jan 31, 2016 |
| Title |
ED479_9189_MALE_rep3 |
| Sample type |
SRA |
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| Source name |
whole animal
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| Organism |
Drosophila melanogaster |
| Characteristics |
developemntal stage: adult
Sex: MALE
drosdel id: Df(2L)ED479
bloomington stock center id: 9189
genetic background: OregonR/w1118
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| Treatment protocol |
S2R+ cells were bathed with ~500 bp double strand RNA for knockdown. The dsRNA treatments followed the DRSC protocol (http://www.flyrnai.org/DRSC-PRR.html). Length of incubation was 1 day.
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| Growth protocol |
Drosophila melanogaster were raised at 25 oC on the standard yeast/cornmeal medium (Fly Facility, University of Cambridge, UK) for w1118 isogenic flies, and on the standard fly agar (Bloomington Drosophila Stock Center, Indiana University, IN) for w1118/OregonR hybrid flies. Virgin w1118 or ModENCODE OregonR females were crossed to DrosDel strain males and the non-balancer flies were collected and aged for 3-5 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the hybrid background flies, single, day 3-5 adult male or female flies were partially crushed, stored in 100ul of RNAlater (Life Technologies, Grand Island, NY), and frozen at - 80oC for long-term storage until RNA preparation. RNA was isolated from each genotype in biological triplicates. We used Mini-BeadBeater 96 (Biospec Products, Bartlesville, OK) for the homogenization of flies. Approximately 100ul of 1mm glass beads (Biospec Products, Bartlesville, OK) were added to the flies in RNAlater in 1ml Axygen 96 well plate (Corning, Union City, CA). We processed plates 3 x 1min with a 2min rest on ice between each homogenization. We added 600ul of RLT buffer (Qiagen, Valencia, CA) to each well to dilute the RNAlater solution. Total RNA was isolated with RNeasy 96 kits (Qiagen, Valencia, CA) according to manufacturer’s handbook (Protocol for Isolation of Total RNA from Animal Cells using spin technology, Cat#19504). The amount of total RNA extract was measured using Quant-iT RiboGreen (Life Technologies, Grand Island, NY).
We mixed 400ng of total RNA in 50ul of nuclease-free water with 50ul of 2:5 dilution of Dynabeads Oligo(dT)25 (Life Technologies, Grand Island, NY) that we pre-rinsed and diluted with Binding Buffer (20mM Tris-HCl pH7.5, 1.0M LiCl, 2mM EDTA). We heated the mixture to 65oC for 5min in a thermocycler, and cooled down on ice for 1min. After 15min of incubation at room temperature, we collected the beads with a magnetic stand, and rinsed with 200ul Washing Buffer (10mM Tris-HCl pH7.5, 0.15M LiCl, 1mM EDTA) for 1min at 1,000 rpm (Thermomixer, Eppendorf, Hauppauge, NY). We collected the beads again with a magnetic stand, and eluted with 50ul of 10mM Tris-HCl pH7.5 at 80oC for 2min. We rebound the eluate to the beads by incubating with 50ul Binding buffer, and rinsed with 200ul Washing Buffer as above. We eluted and fragmented the poly A+ RNA with 16ul of Fragmentation Buffer that contained 1:4 dilution of 5X First Strand Buffer from Protoscript II (New England BioLabs, Ipswich, MA), 500ng of random primers (Life Technologies, Grand Island, NY), and 20pg of ERCC spike-ins Pool 78A or 78B (Zook et al., 2012. PLOS One) at 9 oC for 6min. The beads were removed using a magnetic stand, we reverse transcribed with10units of SuperRase-in (Life Technologies, Grand Island, NY), 100units of Protoscript II reverse transcriptase, 5mM DTT, and 625 uM dNTPs (Enzymatics, Beverly, MA.). We used a thermocycler set at 25oC for 10min; 42oC for 50min; 70oC for 15min. We cleaned-up the DNA-RNA hybrids with 1.9 volumes of MagNA beads (Rohland and Reich, 2012. Genome Research), and mixed with 0.85 volumes of ethanol and we bound DNA-RNA hybrid to the beads at room temperature for 15min. We collected beads on a magnetic stand and rinsed with 200ul of 80% ethanol twice. After air-drying for 5min, we eluted the beads with 16ul of Elution Buffer. For the second strand synthesis, we added 2.5 units of RNase H (Enzymatics, Beverly, MA.) and 10 units of DNA polymerase I in 1X Blue Buffer (Enzymatics, Beverly, MA.), 10mM DTT, 0.5mM each dATP, dCTP, and dGTP (Enzymatics, Beverly, MA.) and 1mM dUTP (ThermoFisher Scientific, Waltham, MA. 1 mM final) and incubated for 5 hours at 16oC. We cleaned-up DNA products MagNA beads and eluted as above but we used bound beads for the next purification steps. Purified double-stranded DNA was subjected to end-repair with NEBNext End Repair Module (New England BioLabs) as per manufacturer’s protocol for 20ul samples. We replenished beads with 1.9 volume of XP buffer (Wang et al., PLOS One. 2011) and cleaned up as above. For adapter ligation, we performed adenylation on blunt-ended DNA by adding 2.5 units of Klenow 3’-5’ exo (Enzymatics, Beverly, MA.) and incubation at 37oC for 30min in 1X Blue Buffer. We again used 1.9 volume of XP buffer to clean up as above. We eluted DNA with 10ul of Elution buffer, and added 1ul of one of 24 differently barcoded adapters from the TruSeq v2 kit (Illumina, San Diego, CA) for 24ul ligation reactions with T4 DNA ligase (Rapid) (Enzymatics, Beverly, MA.) for 20min at 20oC. We stopped the ligation by adding ⅓ volume of 0.03M EDTA, and cleaned up with 24ul of XP buffer as above. After eluting with 30ul Elution buffer, we cleaned up again with 1 volume of XP buffer add brought samples to a 12ul final volume. We digested dUTP incorporated strands of DNA with 5 units of Uracil DNA Glycosylase (New England BioLabs) for 30min at 37oC. Then we amplified with 1.5ul of P5 and P7 primers (Integrated DNA Technology, Coralville, Iowa) with KAPA HiFi HotStart DNA polymerase (KAPA Biosystems, Wilmington, MA) in 30ul reactions. We used the following PCR parameters: 98oC for 45 sec, 14 X 98 oC for 15sec; 60oC for 30sec; 72 oC for 30 sec, followed by 72oC for 5min. We cleaned up amplified DNA with MagNA beads. Libraries were quantified with Quant-iT PicoGreen (Life Technologies), and pooled to be sequenced in Illumina HiSeq 2000 (Illumina, San Diego, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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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 |
Oct 12, 2015 |
| 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) |
| GSE73920 |
Expression profiling individual DrosDel flies heterozygous for deletions of chromosome 2L in a hybrid background |
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| Relations |
| Reanalyzed by |
GSM3277077 |
| SRA |
SRX1329372 |
| BioSample |
SAMN04160623 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1906128_ED479_9189_MALE_rep3_cufflinks.txt.gz |
484.4 Kb |
(ftp)(http) |
TXT |
| GSM1906128_ED479_9189_MALE_rep3_htseq.txt.gz |
56.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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