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| Status |
Public on Jun 13, 2017 |
| Title |
Housefly_control rep1 |
| Sample type |
SRA |
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| Source name |
whole body
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| Organism |
Musca domestica |
| Characteristics |
tissue: whole body
strain: MdSGHV
treatment: PBS-treated
age: Adult female
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| Treatment protocol |
Cohorts of synchronously infected houseflies were produced by injecting newly emerged females with filter-sterilized viremic SG homogenates, a treatment that guarantees symptomatic infection in 100% of the injected flies. Control flies were injected with sterile PBS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At 48 h post-challenge, PBS- and virus-injected females were placed individually in tubes containing aliquots of 1 ml of Tri-Reagent (Sigma-Aldrich). Each sample was homogenized by adding ~ 20 zirconium beads (BioSpec Products, Bartlesville, OK) followed by vigorous shaking (30s) in a bead-homogenizer (FastPrep® Instrument, Qbiogene, Carlsbad, CA). Total RNAs were extracted according to the TriReagent protocol. Ethanol-precipitated RNA pellets were suspended in 100 µl DEPC-treated water and treated with RNase-free DNase (Qiagen). The isopropanol-precipitated total RNAs were re-extracted using SV Total RNA Isolation Kit (Promega, Fitchburg, WI, USA). RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer and the Agilent 2100 Bioanalyzer.
Preparation and sequencing of RNA libraries were performed by ICBR/UF (Gainesville, FL USA) according to the manufacturer’s instructions (Illumina, Inc.) using the NextSeq500 platform. Briefly, the mRNA was enriched from 1 µg of total RNA per sample using oligo-dT attached magnetic beads and then subjected to thermal fragmentation using the elute, prime, and fragmentation mix from Illumina TruSeqTM v2 RNA sample preparation kit. RNA fragments were then converted to double-stranded (ds)-cDNA using reverse transcriptase and random primers provided in TruSeq RNA sample preparation kit. The ds- cDNA fragments were end-repaired by enzymatic polishing with T4 DNA polymerase and E. coli DNA polymerase I Klenow fragment. A single non-templated dA-tail was added to 3’-end of the repaired fragments and then ligated to NEB adaptors (NEBNext®Utra RNA library preparation kit). The required fragments were purified by AMPure beads (Agencourt; PN A63881) and enriched by PCR amplification. The amplified libraries were purified and quantified using the Agilent DNA high-sensitivity kit on an Agilent 2100 Bioanalyzer and qPCR.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
The flies came from the USDA Center for Medical Agricultural and Veterinary Entomology, Gainesville, FL, USA
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Several quality measures were performed using the web interface/software program Galaxy’s FastQC command to check the data quality of the reads and detect any potential errors introduced during sequencing or library preparation. The Cutadapt software program was used to remove low quality reads from the final dataset. Reads with a Phred-like score of <40 and bases with a score <20 were excluded. The genes or transcripts of M. domestica (38,323 sequences) from NCBI were used as reference sequences for RNA-Seq analysis using the Bowtie2 mapper.
ICBR scripts along with SAMtools and the DESeq, EdgeR, and Bayseq (DEB) computational applications were applied to generate the gene expression data, including fold changes and p-values, to remove duplicates or artifacts from PCR, and choose uniquely mapped reads for gene expression analysis. Digital gene expression was assessed by counting the number of transcripts that mapped to a gene sequence
Genome_build: mus_refseq-38323.fasta
Supplementary_files_format_and_content: tab-delimited text files include the mapped read counts for each Sample
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| Submission date |
Oct 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
FAHONG YU |
| E-mail(s) |
FYU@ufl.edu
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| Phone |
3522738064
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| Organization name |
UNIVERSITY OF FLORIDA
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| Department |
ICBR
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| Street address |
2033 Mowry Rd
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| City |
GAINESVILLE |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platform ID |
GPL22576 |
| Series (1) |
| GSE88939 |
Impacts of the Hytrosavirus on the Transcriptome of the Housefly, Musca domestica |
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| Relations |
| BioSample |
SAMN05930785 |
| SRA |
SRX2255216 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2355585_C3_ind2_count.txt.gz |
112.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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