biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsBrian OliverRNA-seq of coding RNADrosophila simulansDrosophila simulanshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-31302We sequenced dissected ovaries and testes (with reproductive tracts) as well as female and male carcasses in two species of Drosophila in order to validate gene predictions from the ModENCODE project. Comparison of dissected reproductive tracts and remaining carcasses between D. simulans and D. pseudoobscurabiostudies-arrayexpressLibrary Construction - Total RNA was extracted from a pool of 30 carcasses using Trizol (Invitrogen, Carlsbad, CA). Libraries were were created using the standard TruSeq Low-Throughput RNA sample protocol (Part# 15008136 Rev. A, November 2010) (Illumina, San Diego, CA). The entire protocol was carried out in PCR tubes and all locations in the protocol where suggested incubation time was 2 or 5 minutes, 5 minutes was used. The sample showed an overabundance of primer-dimers when analyzed by BioAnalyzer (Agilent, Santa Clara, CA) after completion of the protocol, so it was run through 2 subsequent rounds of purification using Ampure XP beads (Beckman Coulter Genomics, Danvers, MA) and the following protocol: 54 ul of Ampure XP beads were added to the sample followed by pipetting up and down 10 times to mix. The sample was incubated at room temp for 5 mins before being placed on an Agencourt SPRIPlate 96 (Beckman Coulter Genomics, Danvers, MA) for 5 mins. The cleared solution was apirated and discarded before adding 200 ul of freshly prepared 70% EtOH and incubating for 30 seconds before removing EtOH (this EtOH was repeated for a total of 2 washes). The beads were allowed to dry for 5 mins on plate with open tubes before transfering them off of the Spriplate and adding 30 ul Tru-Seq resuspension buffer and pipetting up and down 10 times to mix. Finally the samples were placed on the Spriplate for 5 mins to elute the DNA, which was transfered to a new tube.Sample Treatment - Aged flies were snap frozen on dry ice and immediately dissected of their gonads and associated reproductive tract in 1X PBS. The reproductive tracts and remaining carcasses were placed in 1.5 ml microfuge tubes containing 1X PBS and snap frozen on dry ice before being placed at -80 degrees C until RNA extraction.Library Construction - Total RNA was extracted from a pool of 60 dissected reproductive tracts using Trizol (Invitrogen, Carlsbad, CA). Libraries were were created using the standard TruSeq Low-Throughput RNA sample protocol (Part# 15008136 Rev. A, November 2010) (Illumina, San Diego, CA). The entire protocol was carried out in PCR tubes and all locations in the protocol where suggested incubation time was 2 or 5 minutes, 5 minutes was used. The sample showed an overabundance of primer-dimers when analyzed by BioAnalyzer (Agilent, Santa Clara, CA) after completion of the protocol, so it was run through 2 subsequent rounds of purification using Ampure XP beads (Beckman Coulter Genomics, Danvers, MA) and the following protocol: 54 ul of Ampure XP beads were added to the sample followed by pipetting up and down 10 times to mix. The sample was incubated at room temp for 5 mins before being placed on an Agencourt SPRIPlate 96 (Beckman Coulter Genomics, Danvers, MA) for 5 mins. The cleared solution was apirated and discarded before adding 200 ul of freshly prepared 70% EtOH and incubating for 30 seconds before removing EtOH (this EtOH was repeated for a total of 2 washes). The beads were allowed to dry for 5 mins on plate with open tubes before transfering them off of the Spriplate and adding 30 ul Tru-Seq resuspension buffer and pipetting up and down 10 times to mix. Finally the samples were placed on the Spriplate for 5 mins to elute the DNA, which was transfered to a new tube.Library Construction - Total RNA was extracted from a pool of 30 carcasses using Trizol (Invitrogen, Carlsbad, CA). mRNA was extracted using the QIAGEN Oligotex kit (Qiagen, Valencia, CA). Illumina library preparation was as follows. We fragmented mRNA using alkaline hydrolysis. First, 9 ul of 100 ng of mRNA and 1 ul of 10X fragmentation buffer (Ambion, Austin, Texas.) was incubated at 70C for 5 minutes to fragment mRNA. One microliter of Stop Buffer (Ambion, Austin, Texas) was added and samples were placed on ice. Fragments were precipitated with 1 ul 3 M NaOAC (pH 5.2), 2 ul glycogen (5 ug/ul , Ambion, Austin, Texas), and 30 ul 100% EtOH and then incubated for 30 minutes at -80 C. Fragmented RNA was pelleted at 14000 rpm with a microcentrifuge at 4C. The pellet was washed with 70% EtOH, air dried and resuspended with 10.5 ul RNase free H2O. Fragmented mRNA was reverse transcribed to create cDNA. One microliter of random hexamer primers (3ug/ul, Invitrogen, Carlsbad, California) was placed with 10.5 ul of fragmented mRNA, then samples were incubated at 65C for 5 minutes and placed on ice. Four microliters 5X first strand buffer, 2 ul 100 mM DTT, 1 ul dNTP, and 0.5 ul RNaseOUT were added to the mRNA and samples were incubated at 25C for 2 minutes. One microliter of SuperScript II (200 U/ul, Invitrogen, Carlsbad, California) was added and samples were incubated under the following conditions: 25C-10 minutes, 42C-50 minutes, 70C-15 minutes. Samples were then placed on ice. Second strand cDNA was synthesized by adding 61ul H2O to the first strand mix, 10 ul 10X second strand buffer (500 mM Tris-HCl pH 7.8, 50 mM MgCl2, 10 mM DTT), and 3 ul dNTP (10 mM), mixed, incubated on ice for 5 minutes, then 1 ul RNaseH (2U/ul, Invitrogen, Carlsbad, California) and 5 ul DNA Pol I (10 U/ul, Invitrogen, Carlsbad, California) were added. Samples were incubated at 16C for 2.5 hours and DNA products purified using a QIAquick spin column and following manufacture directions and eluted with 30 ul. cDNA fragment libraries were prepared for sequencing. The ends of cDNA fragments were repaired using 30 ul of DNA, 45 ul H2O, 10 ul T4 DNA ligase buffer with 10 mM ATP, 4 ul dNTP mix (10 mM), 5 ul T4 DNA polymerase (3 U/ul), 1 ul Klenow DNA polymerase (5U/ul), and 5 ul T4 PNK(10 U/ul). Samples were incubated for 30 minutes at 20C, purified with QIAquick PCR spin columns, and eluted with 32 ul of Buffer EB. A single 'A' base was added to the ends of cDNA by using 5 ul Klenow buffer, 10 ul dATP (1 mM) and 3 ul Klenow 3' to 5' exonuclease (5 U/ul). Samples were incubated at 37C for 30 minutes, purified with a QIAquick MiniElute column, and eluted with 19ul of EB solution. Paired-end adaptor oligonucleotdies were ligated to A overhang fragments as follows. Overhang fragments were combined with 25 ul DNA ligase buffer, 1 ul adaptor olio mix, and 5 ul DNA ligase (1U/ul). Samples were incubated at room temperature for 15 minutes, purified with a QIAquick MiniElute column, and eluted with 10 ul of buffer EB. cDNA templates were sized selected by gel purification then amplified by PCR using oligo-specific primers. Samples were loaded into a 2% agarose gel with 1X TAE buffer and run for 50 minutes with a 100 bp ladder. The gel was stained with ethidium bromide and bands were visualized under UV fluorescence. For each sample, a gel slice was cut at the 200 (+/- 25) bp region, purified with a QIAquick gel extraction kit, and eluted with 30 ul buffer EB. Purified cDNA was amplified in a 50 ul reaction using 25 ul Phusion Mix (Phusion polymerase, dNTPs, and buffer), 1 ul PE primer 1.1, 1 ul PE primer 2.1, and 23 ul template along with the following cycling conditions: initial denaturation 98C-30 sec, cycle denaturation 98C-10 sec, anneal 65C-30 sec, and extend 72C -30 sec with 15X cycles and a final extension of 72C for 5 minutes followed by a hold at 4C. The amplified product was purified using a QIAquick column and eluted with 30 ul buffer EB.Growth Protocol - Virgins were collected under CO2 anestesia and aged 5-8 days on standard cornmeal medium at 25 degrees C.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesNicolas MattiuzzoJohn MaloneBrian OliverCarlo ArtieriData Transformation - Reads were either generated as 76 or 101 bp paired-end. However, all 101 bp reads were trimmed to 76 bp due to low quality on the 3' end. Trimmed 76 bp reads were used for subsequent analyses. Trimmed reads that passed the Illumina filter were mapped with Tophat 1.2.0. The species-specific reference sequence (FlyBase release 2 in the case of Drosophila pseudoobscura and FlyBase release 1 in the case of Drosophila simulans) was used to build a reference index for mapping. The reference index was created using the bowtie-build function (bowtie version 0.12.7) with default parameters and reads were mapped to this reference using the Tophat parameters -g 1 -F 0 -r 148 -i 42 -solexa1.3-quals.falseRNA-Seq of Gonads and Carcasses in D. simulans and D. pseudoobscuraWe sequenced dissected ovaries and testes (with reproductive tracts) as well as female and male carcasses in two species of Drosophila in order to validate gene predictions from the ModENCODE project. Comparison of dissected reproductive tracts and remaining carcasses between D. simulans and D. pseudoobscura2011-08-26T00:00:00Z2023-08-18T22:47:57.073Z2022-03-04T14:53:59.553ZE-GEOD-31302SRP007818EFO_0003738