Long-read based assembly and annotation of a Drosophila simulans genome
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PROVIDER: PRJEB28741 | ENA |
REPOSITORIES: ENA
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Project description:High-throughput sequencing of Drosophila pseudoobscura and Drosophila simulans small RNAs. ~18-26nt RNAs were isolated from total RNA using PAGE, ligation to adapters requires 5' monophosphate and 3' OH. Small RNAs were cloned from Drosophila pseudoobscura (heads and pooled 0-12 and 12-24 hour embryos) and Drosophila simulans (pooled 0-12 and 12-24 hour embryos). Sequencing was performed using the Illumina 1G platform. Following removal of 3' linker sequences, the clipped sequences longer than 18 nt were kept.
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Project description:Y chromosome from different Drosophila simulans were introgressed into the same genetic background. Strains showing distinct sex-ratio distortion were clustered according to the ratio of males and females observed in the progeny. Strains showing disproportionally high number of female flies in the progeny were contrasted with strains displaying the proportion of male and females flies close to 1:1. In addition, interspecific Y chromosome from Drosophila sechellia (Ysec) was compared with D. simulans Y chromosomes. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). A minimum of 2 biological replicas was used, however this number varied according to the hybridization design (See 'Branco et. al, Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans' for details of the hybridization design). Microarrays were ~18,000-feature cDNA arrays spotted with D. melanogaster cDNA PCR products. We have recently annotated these probes using the D. simulans genome. Possible biases introduced by using a D. melanogaster platform for a D. simulans genome are minimized because the probed genomes are essentially identical except for the Y chromosomes. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma.
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