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| Status |
Public on Apr 03, 2012 |
| Title |
Wholeflies_ED499_Female_R2_SOLiD_a |
| Sample type |
SRA |
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| Source name |
Whole flies, 5 day mated adults
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| Organism |
Drosophila melanogaster |
| Characteristics |
gender: Female
strain: Df(2L)ED499/+
tissue: whole flies
developmental stage: 5 day old mated adults
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| Treatment protocol |
We collected replicates for experiments by outcrossing the balancer chromosome with the original wild-type stock (w1118) as follows; 18-24 vials were setup with 15 less than 8 day old virgin w1118 females paired with 5 less than 8 day old males of a particular deficiency line per vial. Df/+ female and males were placed together in vials at less than 60 per vial and then aged for five days. On day five, flies were anesthetized with CO2, sorted into males and females, then placed into 1.5 ml Eppendorf tubes and flash frozen on dry ice. All flies aged together for 5 days constituted a replicate. Flash frozen flies were stored in -80°C before RNA extraction.
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| Growth protocol |
Flies were grown under constant light, temperature, and humidity (25°C; 60% relative humidity) on San Diego Stock Center cornmeal media (DSSC, 2008)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The spike-in control mixtures were designed to assess technical performance of a typical gene expression experiment examining enrichment between two conditions. These mixtures consist of the 96 distinct control RNAs from the ERCC (External RNA Control Consortium) project. The RNAs were synthesized, purified, and mixed at NIST. The mixtures (Mix 1 and Mix 2) contain 3 subsets of 32 RNAs: one subset at equal abundance in Mix 1 and Mix 2, representing a “no-change” control; one subset at 1.5 fold enrichment in Mix 1 relative to Mix 2; and another subset at 1.5 fold enrichment in Mix 2 relative to Mix 1. Each subset of 32 control RNAs was designed to span a concentration dynamic range of 220, or 1048576:1. The control IDs and concentrations in Mix 1 and Mix 2 are annotated in the supplementary file linked to the GSE31401 record.
ERCC Spike-in Mix 1 was combined with the diploid control mRNA samples (female and male w1118) and ERCC Spike-in Mix 2 was combined with mRNA from all other DrosDel samples at about 10% concentration the total quantity of mRNA.
Approximately 150-200 ng of polyA selected mRNA was converted to cDNA, using the SOLiDTM Total RNA-Seq Kit PN 4445374 and accompanying kit protocol PN4452437 Rev. A (Life Technologies, Carlsbad, CA). RNA was enzymatically fragmented with RNase III using stated protocol methods and kit supplied components. The fragmented RNA was then cleaned and concentrated using the RiboMinusTM Concentration Module (Invitrogen Corporation, Carlsbad, CA) and eluted in 12 u L of nuclease-free water. The fragmented RNA was then assessed for size distribution and concentration on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA) and the Qubit® RNA assay (Invitrogen Corporation, Carlsbad, CA), respectively. Fragment distributions were found to have a mean distribution of approximately 150 nts. In preparation for library construction the RNA needed to be sufficiently concentrated such that less than or equal to 3u L of fragmented RNA contained 50 ng. Concentration was performed in a SpeedVac® Concentrator (Savant Instruments Inc., Holbrook, NY) under vacuum and medium heat for approximately 10 minutes. Each dried fragmented RNA sample was then rehydrated to a final concentration of 17 ng/u L in nuclease-free water. Hybridization and ligation of partial P1 adaptor and internal adaptor, at the 5' or 3' end respectively, was performed using concentrated fragmented RNA and kit supplied adaptors and hybridization mixtures. Reverse transcription was then performed as directed by protocol with kit supplied ArrayScriptTM Reverse Transcriptase (Ambion Inc., Austin, TX) and necessary reaction components. Resulting cDNA was then purified using the MinElute® PCR Purification Kit (Qiagen Inc., Valencia, CA) and eluted in 10 u L of Buffer EB supplied by the purification kit. cDNA libraries were then size selected for 150-250 nts fragments that represent cDNA with target sequence and 5' and 3' SOLiDTM sequencing adaptors. The cDNA was run on a Novex® 6% TBE-Urea Gel 1.0 mM (Invitrogen Corporation, Carlsbad, CA) using the XCell SureLockTM Mini-Cell system (Invitrogen Corporation, Carlsbad, CA) to separate the constituent sizes of cDNA. Samples and ladder were loaded on to gel, separated by a lane to minimize cross contamination, and run at 180 V for approximately 30 minutes. The gels were then stained in 50 u L of 1X SYBR Gold nucleic acid gel stain (Invitrogen Corporation, Carlsbad, CA) solution prepared with 1X TBE Running Buffer. Gel and stain were agitated in the dark at room temperature for 10 minutes then imaged with blue-light transilluminator. Using a clean razor blade for each cut the targeted size range of 150-250 nts was excised and, with vertical orientation maintained, vertically cut into four ~ 1mm x 6mm slices. Two of the four slices were used in amplification while the remaining two slices were stored for future use, if necessary, at -20°C. In-gel Polymerase Chain Reaction (PCR) amplification was then performed using excised gel slices and reaction components and primers supplied by the kit. The primers supplied extend and complete the P1 adaptor at the 5' end while primers containing unique barcodes were used to add a unique barcode and P2 adaptor to the 3' end. All samples were uniquely barcoded using the SOLiDTM RNA Barcoding Kit, Modules 17-32 (Life Technologies, Carlsbad, CA). Samples were amplified with AmpliTaq® DNA Polymerase (Applied Biosystems, Carlsbad, CA) using thermal cycling parameters designated by the kit protocol on a PTC-200 DNA Engine (MJ Research, Watertown, MA) for 15 cycles to avoid overamplification. Amplified samples were purified using the PureLink PCR Micro Kit (Invitrogen, Carlsbad, CA). Samples were eluted in 20 u L of Elution Buffer supplied with the purification kit. Following barcoding and amplification, completed cDNA libraries were assessed for size distribution and yield on the 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA). A smear analysis was performed and confirmed <20% of resulting cDNA was smaller than 200 nucleotides and therefore appropriate for templated bead preparation. Individual cDNA libraries were quantified using quantitative PCR (qPCR) on a 7900 HT Real-Time PCR System (Life Technologies, Carlsbad, CA) and pooled with other barcoded samples to create an equimolar pool. Pooled libraries were quantified using qPCR to confirm final molar concentration for use in templated bead preparation. Templated beads were prepared with emulsion polymerase chain reaction (ePCR) using the Applied Biosystems EZ Bead System (Life Technologies, Carlsbad, CA). Two library titration points, 0.4 and 0.8 pM, were initially used for work flow analysis (WFA) to determine input for full ePCR. Upon completion of WFA run on the SOLiDTM v4 instrument it was determined that a 0.6 pM titration point would be used in templated bead preparation to achieve maximum yield of high quality (greather than or equal to 77% On-Axis, less than or equal to 7% Noise to Signal) P2 enriched beads. Samples were run in duplicate in quads on two flow cells of a SOLiDTM v4 platform using ToP chemistry to generate paired-end reads of 50 base pair forward, 35 base pair reverse and a 10 base pair barcode.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
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| Description |
Flowcell 1; Same poly A mRNA prep used for microarray hybridization
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| Data processing |
High quality reads were matched to individual samples based on the 10 bp bar code tag and using default parameters in SOLiD Experimental Tracking Software (SETS) v4.0.1. We used only the forward read and trimmed these reads from 50 to 36 bp based on analysis of read quality. Trimmed reads were mapped with Bowtie. The dm3 from the UCSC Genome Browser which corresponds to the April 2006 Drosophila melanogaster draft assembly from the Berkeley Drosophila Genome Project (Release 5) plus sequences of the 96 synthetic RNAs were used to build a reference index for mapping colorspace data. The reference index was created using the bowtie-build function with default parameters for colorspace. Reads were mapped using -v 2 -m 1 in Bowtie v. 0.12.7 and then unique mapping sequences that mapped to spike-in controls and Flybase gene models were counted using the union mode in HTSeq. Technical replicates for each sample were highly comparable and we pooled reads between the two flow cells for each sample.
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| Submission date |
Aug 16, 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 |
GPL14601 |
| Series (2) |
| GSE31401 |
RNA-Seq on SOLiD platform of DrosDel deficiency and w1118 flies |
| GSE31407 |
Drosophila DrosDel deficiency and diploid control flies |
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| Relations |
| SRA |
SRX092018 |
| BioSample |
SAMN00709516 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM778882_F9274_bowtiemap_v2m1_F3read_trim36.sam.gz |
46.7 Mb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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