|
| Status |
Public on May 17, 2017 |
| Title |
P1-2 |
| Sample type |
SRA |
| |
|
| Source name |
midgut
|
| Organism |
Aedes aegypti |
| Characteristics |
feeding protocol: Uninfected saline meal (PBS)
time (postfeeding day of midgut sampling): 1 day after feeding
tissue: pool of 15 dissected midguts
|
| Treatment protocol |
CHIKV infected as treatment compared against uninfected Ae. aegypti HWE mosquitoes; protein meal (BSA) or saline meal (PBS) fed compared with sugarfed Ae. aegypti HWE mosquitoes
|
| Growth protocol |
One week-old females of Ae. aegypti mosquitoes of the Higgs White Eye (HWE) strain were deprived of sugar for 24 h, and then fed for 1 h with a mixture of equal amount of CHIKV-infected cell culture or non-infected cell culture mixture (control) and 40% BSA or 2xPBS solution at 37°C using a single glass feeder per carton. Fully engorged females were selected and reared in 1.9 L (64 oz.) cartons and offered raisins and water until the further analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 15 midguts (= 1 sample) using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA samples were DNase-treated to remove potential contamination from genomic DNA. RNA purity and concentration were analyzed with a NanoDrop 1000 v 1.3.2 (Thermo Scientific, Wilmington, DE). The absence of RNA degradation was initially assessed by electrophoresis of 1 μg of RNA on a 1.0% agarose gel. Thereafter, the quality of each RNA sample was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with an RNA Nano Chip.
mRNA samples were further prepared for RNA-Seq analysis using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). First-strand cDNA synthesis produced single-stranded DNA copies from the fragmented RNA by reverse transcription. After second-strand cDNA synthesis, the double-stranded DNA underwent end-repair and the 3’ends were adenylated. Finally, universal adapters were ligated to the cDNA fragments and PCR was performed to produce the final sequencing library. These template molecules were used for cluster generation and sequencing on the Illumina NextSeq 500 instrument with multiple lanes and paired-end read (2 x 75 bases). Each treatment was analyzed as three independent biological replicates.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
The raw sequences (fastq) were subjected to quality check by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The adapter removal and quality trimming of the raw reads were done using fqtrim (https://ccb.jhu.edu/software/fqtrim/). Read shorter than 30 nucleotides and quality score less than 25 were excluded from further analysis.
The trimmed reads generated were subjected to mapping to the Aedes aegypti reference genome AaegL3 using Hisat2 mapper
FeatureCounts was used to quantify transcript abundance in each sample using the gene annotation AaegL3.3 obtained from VectorBase (https://www.vectorbase.org/organisms/aedes-aegypti).
Differential expression (DE) analysis between sample groups were performed by edgeRrobust.
Genome_build: AaegL3
Supplementary_files_format_and_content: The processed data is read counts for each gene for all the 27 samples in a text format file.
|
| |
|
| Submission date |
Feb 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander Franz |
| E-mail(s) |
franza@missouri.edu
|
| Organization name |
University of Missouri
|
| Department |
Department of Veterinary Pathobiology
|
| Street address |
303 Connaway Hall
|
| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65211 |
| Country |
USA |
| |
|
| Platform ID |
GPL22030 |
| Series (1) |
| GSE95378 |
The midgut transcriptome of Aedes aegypti fed with saline or protein meals containing chikungunya virus reveals genes potentially involved in viral midgut escape |
|
| Relations |
| SRA |
SRX2590893 |
| BioSample |
SAMN06466333 |
