Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MRNA sequence data of individual Drosophila melanogaster male and female flies from 16 Drosophila Genetic Reference Panel lines reared in replicated environments

ABSTRACT: Our primary objective was to characterize the amount of variation in transcript abundance among individual flies with identical genotypes. We also wanted to determine which analysis methods would be optimal for RNA-Seq data. To meet these objectives, we performed transcriptional profiling of whole adult individuals from 16 Drosophila Genetic Reference Panel (DGRP) lines. We quantified differential expression among genotypes, environments, and sexes. We randomly chose 16 DGRP lines for this experiment: DGRP-93, DGRP-229, DGRP-320, DGRP-352, DGRP-370, DGRP-563, DGRP-630, DGRP-703, DGRP-761, DGRP-787, DGRP-790, DGRP-804, DGRP-812, DGRP-822, DGRP-850, and DGRP-900. We collected 8 virgin male and 8 virgin female flies from the 16 DGRP genotypes in three replicated environments to produce RNA sequence profiles. We controlled the environmental conditions in the following ways. We seeded the fly cultures with 5 male and 5 female parents. We reared the progeny in a single incubator on standard Drosophila food (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) at 25Â°C, 60% humidity, and a 12:12-hour light:dark cycle. We collected and maintained male and female virgins at 20 flies to a same-sex vial for four days prior to RNA extraction to control for social exposure. Flies were frozen for RNA extraction at the same circadian time (1:00 pm) in 96-well plates. PolyA RNA stranded libraries were prepared by modifying an existing protocol. ERCC (External RNA Controls Consortium, SRM2374, beta version, pools 78A/78B) sequences were added during the library preparation as a control. For some samples >1 library was generated to check technical variation. We performed multiplexed single-end 76 bp sequencing on an Illumina HiSeq2000. Reads were mapped to FlyBase release 5 version 57 and release 6 version 01 of the Drosophila melanogaster genome and the ERCC sequences. Mapped reads were counted at the gene level.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Brian Oliver

PROVIDER: E-GEOD-60314 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane. Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 Âµg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 ÂµL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5â-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.

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MRNA sequence data of individual Drosophila melanogaster male and female flies from 16 Drosophila Genetic Reference Panel lines reared in replicated environments

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