GSM2645722: Dmel male TW rep1; Drosophila melanogaster; RNA-Seq - SRA

SRX2876829: GSM2645722: Dmel male TW rep1; Drosophila melanogaster; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 104.8M spots, 21.2G bases, 14Gb downloads

Submitted by: NCBI (GEO)

Study: Transcriptional profiling of adult Drosophila antennae by high-throughput sequencing

PRJNA388757 • SRP108417 • All experiments • All runs

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Background Antennae of fruit flies are the major organs responsible for detecting environmental volatiles, e.g., egg-laying substrates. An adult antenna contains many sensilla full of olfactory sensory neurons, where olfactory receptor (Or) genes are expressed. Each sensory neuron only expresses up to three receptors, making it difficult to estimate expression levels by conventional methods. In this study, we applied Illumina RNA sequencing (RNA-seq) to study the expression levels of Or and other genes in fly antennae. Results RNA from approximately 1,200 pairs of adult antennae from each sex of Drosophila melanogaster was used to obtain the antennal transcriptome of each sex. We detected approximately 12,000 genes expressed in antennae of either sex. The most highly expressed genes included pheromone-binding genes, transmembrane transporter genes, and sensory reception genes. Among the 61 annotated Or genes, we observed 53 and 54 genes (approximately 90%) expressed (fragments per kilobase of exon per million fragments mapped (FPKM)?>?0.05) in male and female antennae, respectively; approximately 25 genes were expressed with FPKM?>?15. Compared to previous studies, which extracted RNA from the whole body or head and used microarrays, antenna-specific transcriptomes obtained by RNA-seq provided more reliable estimates of gene expression levels and revealed many lowly expressed genes. Ninty-one genes, including one odorant-binding protein (Obp) gene and four Or genes, were differentially expressed between male and female antennae. These sexually biased genes were enriched on the X chromosome and showed enrichment in different gene ontology categories for male and female flies. The present and previous data together suggest that a gene family with putative immune response functions is related to pheromone detection and involved in the courtship behavior of male flies. Conclusions Tissue-specific RNA-seq is powerful for detecting lowly expressed genes. Our study provides new insight into the expression of olfactory-related genes in Drosophila antennae.ophila sechellia relies exclusively on the fruits of Morinda citrifolia, which are toxic to most insects, including its sibling species D. melanogaster and D. simulans. Although several odorant binding protein (Obp) genes and olfactory receptor (Or) genes were suggested to be associated with the D. sechellia host shift, a broad view of how chemosensory genes have contributed to this shift is still lacking. We therefore studied the antennal transcriptomes, the main organ responsible for detecting food resource and oviposition, of D. sechellia and its two sibling species. We wanted to know whether gene expression, particularly chemosensory genes, has diverged between D. sechellia and its two sibling species. Using a very stringent definition of differential gene expression, we found 147 genes (including 11 chemosensory genes) were up-regulated while only 81 genes (including 5 chemosensory genes) were down-regulated in D. sechellia. Interestingly, Obp50a exhibited the highest up-regulation, a ~100 fold increase, and Or85c – previously reported to be a larva-specific gene– showed ~20 fold up-regulation in D. sechellia. Furthermore, Ir84a, proposed to be associated with male courtship behavior, is significantly up-regulated in D. sechellia. We also found expression divergence in most of the receptor gene families between D. sechellia and the two sibling species. Our observations suggest that the host shift of D. sechellia is associated with expression profile divergence in all chemosensory gene families and is achieved mostly by up-regulation of chemosensory genes.the evolutionary behaviour of Polycomb group proteins, their recruitment factors and their underlying sequences by performing ChIP-seq analysis in 4-5 different Drosophila species (GSE60428) and HiC analysis in Drosophila melanogaster. We demonstrate an extremely high conservation of Polycomb repressive domains across Drosophila species We validate few cases of PRE divergence that shows that cis-driven PRE evolution is a rare event. We further show that PHO recruitment to Polycomb domains is evolutionarily robust to motif changes and that PRC1 stabilizes binding of its key recruiter Overall design: RNAseq experiments in wild type drosophila antennaes

Sample: Dmel male TW rep1

SAMN07184402 • SRS2246226 • All experiments • All runs

Organism: Drosophila melanogaster

Library:

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: PAIRED

Construction protocol: About 300-700 pairs of fly antennae of each sex from each species were collected for total RNA isolation. The antennae resected each day were preserved in ~ 50-100 ul Trizol (Life Technology) and stored at -80 °C before further processing. Right before the RNA isolation, we spun down antennae preserved in Trizol at the max speed for 3 min at 4 °C and put each sample into one MagNA Lyser Green Beads tube (Roche). The RNA isolation protocol we developed combined steps from conventional Trizol extraction and RNeasy Micro Elute Kit (QIAGEN, Inc.) with some modifications to obtain high yield and quality of total RNA. Fly antennae were disrupted with MagNA Lyser (Roche) at 7000 rpm for 15 seconds each time and repeated for 4-5 times until the tissue was almost invisible. After 15 sec, we cooled down the lysate on ice for 10 sec to prevent RNA degradation. Tissue lysate was transferred to a new RNase free Eppendorf tube. We used about 400 ul Trizol to rinse the beads of each sample and combine this 400 ul Trizol with the tissue lysate for the RNA isolation. Based on the suggestion of the Trizol standard protocol, 200 microliters of chloroform per microliter of tissue lysate were added to the lysate and mixed well by shaking vigorously for 15 sec and set 2-3 min at room temperature. The aqueous phase was separated by centrifuging the lysate at the maximum speed at 4 °C for 15 min and carefully transferred to a new Eppendorf tube after centrifuging. We added 1 volume of 5 µl Carrier RNA (QIAGEN, Inc.) and one microliter of 70% EtOH to the aqueous phase and mixed well by inverting the tubes carefully. To obtain greater amount of RNA, we kept the samples at -80 °C for overnight to aid in RNA precipitation. RNA isolation followed manufacturer’s protocol of the RNeasy Micro Elute Kit with increased volumes (700 ul) of RPE buffer and 80% ethanol to wash RNA. Genomic DNA was removed by applying on-column DNase I treatment at room temperature for 15 min. For paired-end mRNA-seq library preparation, we used Illumina TrueSeq kits. A total of 10 µg total RNA was used for mRNA enrichment by oligo-dT beads followed by cation-catalyzed fragmentation for 4 minutes at 94˚C. The mRNA fragments were then converted into double stranded cDNA by random priming followed by end repair. The fragmented cDNAs from each sex of each species were then ligated to the barcoded paired-end adaptors and subjected to 15 cycles of PCR amplification and purified by Ampure beads (Beckman Agencourt). The absolute concentrations of the libraries were determined by Qubit fluorometry (Invitrogen) and BioAnalyzer High Sensitivity DNA Kit (Agilent). We combined barcoded cDNA from 6 samples and loaded them in 3 sequencing lanes of flow cell, and paired-end 2*100nt sequencing was conducted on Illumina HiSeq2000.

Experiment attributes:

GEO Accession: GSM2645722

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 104.8M spots, 21.2G bases, 14Gb

Run	# of Spots	# of Bases	Size	Published
SRR5638358	104,813,095	21.2G	14Gb	2017-06-05

ID:: 4118620

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