|
| Status |
Public on Sep 12, 2015 |
| Title |
Dmel_infected_Phootrhabdus_30h |
| Sample type |
SRA |
| |
|
| Source name |
whole flies_infected_Photorhabdus
|
| Organism |
Drosophila melanogaster |
| Characteristics |
drosophila (host) strain: OregonR
drosophila (host) age: 4-6 days-old
developmental stage: adult
Sex: 1:1 ratio male/female
sample collection time: 30 h post-infection
# of flies used for extraction: 40
infected with: Photorhabdus luminescens subsp laumondii
pathogen strain: TT01
pathogen age/stage: fresh overnight culture
infection dosage: 500-700 CFUs/fly
|
| Treatment protocol |
Infection Protocol: Nematode infections were carried out using nested 5 mL cups (Solo®) and filter papers (Whatman) that supported 10-15 adult flies per group. A 500-700 μL solution containing symbiotic or axenic Heterorhabditis nematodes was added to each container (100 IJ/fly). Treatments involving sterile water devoid of nematodes were used as negative controls. Infected and control flies were kept at 25°C. A PBS suspension (18.4 nL) containing Photorhabdus bacteria was injected into Drosophila adult flies at the lateral anterior side of the thorax through nano-injection (Nanoject II - Drummond Scientific). The number of Photorhabdus cells delivered into each fly was approximately 500-700 colony-forming units (CFUs). Control samples involved PBS injected flies and kept under the same conditions [PMID 22546901].
|
| Growth protocol |
Drosophila melanogaster: Oregon R adult flies were used for the transcriptomic analyses. Flies were reared on instant Drosophila diet (Formula 4-24 Drosophila medium) supplemented with yeast (Carolina Biological Supply), and maintained at 25°C and a 12:12-h light:dark photoperiodic cycle. Male and female adult flies aged 4–6 days old were used in infection assays with the nematodes and their bacteria [PMID 22546901].
Heterorhabditis bacteriophora (Nematode): The entomopathogenic nematodes Heterorhabditis bacteriophora (strain TT01) were amplified in fourth instar larvae of the wax moth Galleria mellonella using the water trap technique. To confirm lack of Photorhabdus bacteria in Heterorhabditis nematodes (axenic), the worms were homogenized and the lysate was spread on selective media. Fresh IJ worms were collected and prepared through pelleting, washing and re-suspending in sterile distilled water. IJ nematodes carrying or lacking Photorhabdus bacteria were used 1-2 weeks after collection from the water traps. Heterorhabditis numbers were estimated by counting the average nematode density present in ten individual 50 μL drops of water using a stereo-microscope [PMID 22546901].
Photorhabdus luminescens (bacteria): The Gram-negative insect pathogenic bacteria Photorhabdus luminescens subsp. laumondii (strain TT01) were used for fly infections. Bacteria were cultured in Luria-Bertani broth (LB) and incubated for 18-24 h at 30°C. Bacterial cultures were centrifuged at 4°C, pelleted, washed in 1x sterile phosphate-buffered saline (PBS) and re-suspended in PBS. Bacterial density was measured using a NanoDropTM 2000c (Thermo Fisher Scientific) and a 10x serial dilution plating technique [PMID 22546901].
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from 40 adult flies infected with Heterorhabditis axenic nematodes, symbiotic nematodes, Photorhabdus bacteria only as well as from uninfected controls. Samples were collected at 12 and 30 h post infection. Total RNA was extracted using the PrepEase RNA spin kit (Affymetrix) following the manufacturer’s instructions. Briefly, flies were homogenized using sterile plastic pestles and RNA was extracted using a silica-based column system including a DNAse treatment step for 15 min. Total RNA was re-suspended in 40 μL of sterile nuclease-free water. RNA concentration was measured using a Nanodrop. RNA integrity and quality were assessed using a Bioanalyser (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
TT01_30h
|
| Data processing |
Base calls were performed using the built-in Illumina basecaller
Sequenced reads were trimmed for adaptor sequence, and then mapped to D. melanogaster whole genome reference using TopHat v1.4.0
Reads Per Kilobase of gene per Million mapped reads (RPKM) were calculated for each sample.
Differential expression of genes were computed
Genome_build: D. melanogaster BDGP5; GCA_000001215.1
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample
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| |
|
| Submission date |
Sep 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Priti Kumari |
| E-mail(s) |
[email protected]
|
| Phone |
410-706-8353
|
| Organization name |
University Of Maryland Baltimore School Of Medicine
|
| Department |
Institute For Genome Sciences
|
| Street address |
801 W Baltimore St
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21201 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (1) |
| GSE61466 |
RNA-Sequencing analysis of the Drosophila transcriptomic response to infection by entomopathogenic nematodes and their mutualistic bacteria |
|
| Relations |
| BioSample |
SAMN03067441 |
| SRA |
SRX700223 |
