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Sample GSM1060491 Query DataSets for GSM1060491
Status Public on Feb 22, 2013
Title Deug_Embryo_replicate1
Sample type SRA
Source name Embryos
Organism Drosophila eugracilis
Characteristics strain: KB1
stock number: 14026-0451.10
developmental stage: Embryo
Sex: Pooled male and female
Growth protocol Flies were inbred by single pair, full-sib crosses for 10-18 generations (with the exception of D. rhopaloa, which did not tolerate inbreeding). For RNA-seq experiments, animals were reared on cornmeal media with a 12:12 light cycle at 25C in a humidified incubator. Adults were aged 1-4 days, and total RNA was extracted using Trizol from pools of 30 adult males, pools of 30 adult females, and from 200-300 ml of 0-24 hour embryos
Extracted molecule polyA RNA
Extraction protocol mRNA was purified from total RNA using Dynabeads® mRNA Purification Kit (Life tech, catalog number: 610-06). Briefly, 2.5 ug total RNA in 50ul DEPC-treated H2O was denatured at 65°C for 5 minutes to disrupt the secondary structure, and immediately cooled on ice for 1 minute. 100 ul of oligo (dT)25 Dynabeads were washed and resuspended in 50 ul of binding buffer before use. The denatured total RNA was added into prewashed Dynabeads and incubated at room temperature for 5 minutes on a rotary shaker.  mRNA-Bead complex was captured on magnet rack and washed twice with 200 ul wash buffer.  The mRNA was eluted by adding 11 ul of H2O to mRNA-Bead complex followed by heated to 75°C for 2 minutes. Tube was placed on magnet rack immediately for 30 seconds. Supernatant containing the purified mRNA was transferred to a fresh RNAse-free PCR tube while the tube was on magnet rack.
The first strand cDNA was reverse transcribed from poly-A mRNA using random hexamer and SuperScript® First-Strand Synthesis kit (Life Tech, catalog number: 11904-018).  Random hexamer was annealed to mRNA by heating mRNA/random hexamer mixture to at 65°C for 5 minutes then cooled on ice.  8.5 ul of The first strand synthesis reaction mix containing 500 uM dNTP, 20 units RNaseOut, 10 mM DTT, 200 unit Superscript II as added to mRNA/random hexamer.  The first strand cDNA was synthesized by incubation reaction at 25C 10 minutes, 42°C 60 minutes, 70°C 15 minutes and hold at 4°C.  The second strand cDNA was synthesized using DNA polymerase I (life Tech, catalog number: 18010-025).  The second strand cDNA synthesis was incubated at 16°C for 2 hrs in a thermocycler. The double strand cDNA was purified with 1.8X Agencourt AMPure XR beads (Beckman coulter, catalog number: A63882 ).
Double stranded cDNA was constructed into Illumina paired-end libraries according to the manufacturer’s protocol (Illumina Inc.). Double strand cDNA was sheared to fragments of approximately 400 bp with the Covaris S2 or E210 system (Covaris, Inc. Woburn, MA). The setting was 10% Duty cycle, Intensity of 4,200 Cycles per Burst, for 55 seconds. Fragments were processed through DNA End-Repair in 100ul containing sheared DNA, 10ul 10X buffer, 5ul End­Repair Enzyme Mix and H2O (NEBNext End-Repair Module; Cat. No. E6050L) at 20°C for 30 minutes; A-tailing was performed in 50ul containing End-Repaired DNA, 5ul 10X buffer, 3ul Klenow Fragment (NEBNext dA-Tailing Module; Cat. No. E6053L) at 37°C for 30 minutes, each step followed by purification using a QIAquick PCR purification kit (Cat. No. 28106).  Resulting fragments were ligated with Illumina PE adapters and the NEBNext Quick Ligation Module (Cat. No. E6056L).  After ligation, size selection was carried out by using 2% low-melt agarose gel running in 1X TBE. Gel slices were excised from 290bp to 340bp and the size-selected DNA was purified using a Qiagen MinElute gel extraction kit and eluted in 30ul EB buffer.  PCR with Illumina PE 1.0 and 2.0 primers was performed in 25-μl reactions containing 12.5 ul of 2x Phusion High-Fidelity PCR master mix, 2.5ul size-selected fragment DNA, 0.3ul each primer and H2O. The standard thermocycling for PCR was 30 s at 98°C for the initial denaturation followed by 12 cycles of 10 s at 98°C, 30 s at 65°C and 30 s at 72°C and a final extension of 5 min. at 72°C.  1.8X Agencourt® XP® Beads (Beckman Coulter Genomics, Inc.; Cat. No. A63882) were used to purify the PCR products. After Bead purification, PCR products were quantified using PicoGreen (Cat. No. P7589) and their size distribution analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500 (Cat. No. 5067-1506).
From the 10nM final library, 15ul was sequenced on Illumina’s Genome Analyzer IIx or HiSeq 2000 instrument according to the manufacturer’s specifications (specified for each sample in SRA entries). Briefly, cluster generations were performed on an Illumina cluster station. Paired-end sequencing was performed, for 90-125 cycles of sequencing for each mate.  Each library was sequenced in a separate, single flow cell lane.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description Deug_E
Data processing Base calls and quality scores were generated by the Illumina analysis pipeline (Casava 1.4 for GAIIx, and Casava 1.6 for Hiseq 2000).  All reads were trimmed to the same length (75bp) for downstream analysis.
Reads were mapped to their respective genomes using Tophat 2 (v2.0.3), accepting only uniquely-mapped reads.  Parameters supplied to Tophat were:  ‘-g 1 -r 150 --solexa1.3-quals.’  Genome references used for each species were: D.biarmipes (Dbia_1.0), D.bipectinata (Dbip_1.0), D.elegans (Dele_1.0), D.eugracillis (Deug_1.0), D.ficusphila (Dfic_1.0), D.kikkawai (Dkik_1.0), D.rhopaloa (Drho_1.0) and D.takahashi (Dtak_1.0), and were downloaded from NCBI.
We used the UCSC genome browser liftover software (Kent, 2002) to project D. melanogaster annotation coordinates (FlyBase r5.45 and ModENCODE Transcriptome V2) to the reference genome of the fourteen sister species.  After defining orthologous regions for features, we estimated expression of these features in the non-melanogaster species by quantifying reads mapping to them.  This was performed by coordinate intersections with base-level genomic coverage data using BedTools (Quinlan, 2010) on each alignment BAM file.  Splice junctions were quantified from BAM files using the Splicing Analysis Toolkit (Spanki,
Genome_build: dm3, Dbia_1.0, Dbip_1.0, Dele_1.0, Deug_1.0, Dfic_1.0, Dkik_1.0, Drho_1.0, Dtak_1.0
Supplementary_files_format_and_content: Text files (*.rpkm) provide RPKM values for five-prime orthologous coding exons. Additional text files (*.juncs.txt) provide splice junction coverages.
Submission date Jan 07, 2013
Last update date May 15, 2019
Contact name Brian Oliver
Phone 301-204-9463
Organization name NIDDK, NIH
Department LBG
Lab Developmental Genomics
Street address 50 South Drive
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
Platform ID GPL16465
Series (2)
GSE43321 mRNA-Seq of whole adult females and males, and miixed sex embryos, from multiple Drosophila species
GSE44612 Comparative Validation of the D. melanogaster Encyclopedia of DNA Elements Transcript Models
BioSample SAMN02197185
SRA SRX098578

Supplementary file Size Download File type/resource
GSM1060491_Deug_E.juncs.txt.gz 694.3 Kb (ftp)(http) TXT
GSM1060491_Deug_E_R1.all.first.CDS.rpkm.gz 97.0 Kb (ftp)(http) RPKM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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