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| Status |
Public on Dec 05, 2020 |
| Title |
Pool of five virgin female w[1118] D. melanogaster (Wolbachia-free/saline-injected), 24 hours post injection, rep B |
| Sample type |
SRA |
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| Source name |
Pool of 5 adult virgin females
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: w[1118]
treatment: Wolbachia-free
treatment: Injected with phosphate buffered saline
timepoint: 24 hours post injection
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| Treatment protocol |
We established in vivo systemic viral infections in adult Drosophila, using a block design with a time series. Flies with or without Wolbachia (W+/W-), were injected with either virus or saline (SINV+/SINV-), and collected at 6, 24, and 48 hours post-injection (hpi). For each unique condition of W-SINV-time, we generated four biological replicates (A-D), with each replicate consisting of a pool of five virgin females. Specific conditions for generating the fly infection conditions are as follows: five-day-old virgin female Drosophila were anesthetized with CO2 and injected with either: (a) 50 nl sterile PBS, or (b) 50 nl of freshly grown SINV (1010 PFU/mL in PBS) using a nanoinjector (Drummond Scientific). Pools of five flies (representing a single biological replicate) were injected in a randomized order across a five-hour time period, and capillary needles were changed between fly types (Wolbachia-colonized or not) and injection type (PBS or SINV) to avoid cross-contamination. Exact time of injection was recorded, and the pool of five females was placed in a vial containing standard cornmeal-agar medium supplemented with antibiotic-antimycotic (Corning) and a fresh Kimwipe. Subsequently. 6, 24, or 48 hpi flies were flash frozen in liquid nitrogen and stored at -80 °C until further processing.
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| Growth protocol |
Fly stocks were maintained on standard cornmeal-agar medium at 25 °C on a 24-hour light: dark cycle under density-controlled conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from pools of flash frozen flies using TRIzol™ Reagent (Invitrogen) following bead-beating, and according to manufacturer’s instructions. rRNAs and other uncapped RNA species were depleted from RNA samples using the Terminator™ 5´-Phosphate-Dependent Exonuclease (Lucigen).
cDNA libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs) following manufacturer’s recommendations, including a seven minute fragmentation time, 10 cycles of PCR amplification, and use of a specific barcode from the NEBNext® Multiplex Oligos for Illumina® Index Primers Set 1 or 2 (New England Biolabs). Quality and quantity of total RNA, depleted RNA, and final libraries was assessed using a TapeStation 2200 (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Pool of five virgin female w[1118] Drosophila melanogaster (Wolbachia-free/saline-injected), flash frozen 24 hours post injection, replicate B
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| Data processing |
Following demultiplexing, reads were mapped to extracted reference transcripts of either the Drosophila melanogaster reference genome (release 6.16) (dos Santos et al., 2014) or the wMel strain Wolbachia genome (GenBank accession NC_002978.6 (Wu et al., 2004)) using the RSEM v. 1.3.0 (Li and Dewey, 2011) programs ‘rsem-prepare-reference’ and ‘rsem-calculate-expression’, employing the default Bowtie aligner (Langmead and Salzberg, 2012).
Transcript abundance was summarized and imported to R v. 3.3.1 ‘Bug in Your Hair’ (R Core Team, 2014) with tximport v. 1.2.0 (Soneson et al., 2015).
Expression values for each gene were normalized with a TMM normalization in EdgeR v. 3.16.5 (McCarthy et al., 2012; Robinson et al., 2010).
Genome_build: Drosophila melanogaster reference genome (release 6.16) (dos Santos et al., 2014)
Supplementary_files_format_and_content: Text file with TMM normalized expression values per gene found to be expressed in the dataset.
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| Submission date |
Dec 04, 2020 |
| Last update date |
Dec 05, 2020 |
| Contact name |
Amelia Ryan Isis Lindsey |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
University of Minnesota
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| Department |
Department of Entomology
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| Lab |
Cargill Building 250; Plant and Microbial Genomics Institute
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| Street address |
1500 Gortner Ave
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| City |
Saint Paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE162666 |
Wolbachia and virus alter the host transcriptome at the interface of nucleotide metabolism pathways |
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| Relations |
| BioSample |
SAMN17002128 |
| SRA |
SRX9632473 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4956418_1-24-B_TMM.txt.gz |
74.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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