|
| Status |
Public on Jan 08, 2019 |
| Title |
Infection_RNAseq_replicate2 |
| Sample type |
SRA |
| |
|
| Source name |
mosquito midgut
|
| Organism |
Anopheles gambiae |
| Characteristics |
tissue: midgut and salivary gland
Sex: female
time: 7 days post-blood meal
infection status: Infected
replicate: R2
|
| Treatment protocol |
Females were fed through membranes on gametocyte-infected blood from malaria patients. To eliminate transmission-blocking factors, venous blood was collected and the serum was replaced by a non-immune AB serum to avoid transmission of human blocking factors
|
| Growth protocol |
Three- to five-day-old female A.gambiae mosquitoes were sourced from an outbred colony established in 2008 and repeatedly replenished with F1 from wild-caught mosquito females collected in Kou Valley (11°23'14"N, 4°24'42"W), 30 km from Bobo-Dioulasso, south-western Burkina Faso (West Africa). Mosquitoes were maintained under standard insectary conditions (27 ± 2°C, 70 ± 5% relative humidity, 12:12 LD)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Dissection of mosquito midguts and salivary glands was performed in situ on adult females at 7 days post-blood meal. To determine infection levels, mosquito guts were dissected 7 days after blood feeding and were stained with 2% Mercurochrome before microscopic examination. Tissues were maintained in ice-cold Schneider’s insect culture medium (Sigma-Aldrich) and fresh tissues were immediately processed for chromatin and RNA analyses
Total RNA was extracted from fresh mosquito tissues (~25 midguts and ~ 50 salivary glands) using the mirVana™ RNA Isolation Kit (Ambion®) according to the manufacturer protocol and used for mRNA library preparation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Infection_RNAseq_coverage.bw
|
| Data processing |
We mapped reads for various histone modifications and the input to A.gambiae PEST strain genome version 4.3 using Bowtie v2.2.9 with default parameters except for --no-mixed
Reads were trimmed 5 bases from each read 5’ end (--trim5 5). Mapped reads were sorted and deduplicated using SAMtools v.1.6. We applied a quality threshold of 10 in MAPQ score. All libraries were downsampled (SAMtools) to the same number of reads (9M) for further downstream analysis
We conducted peak calling using MACS2 (v.2.1.1) [61] “callpeak” module with -t and -c options and default parameters, except for -g 2.73e8 --keep-dup all -B --SPMR -q 0.01 –nomodel
To quantitative compare histone modification profiles between infected and control mosquito tissues, we used diffReps software. ChIP-seq data for infected and control and the corresponding inputs were provided with -tr, -co, --btr and --bco options. Due to the lack of biological replicates, the statistical test used was G-test (-me gt). Rest of parameters were set as default except for --window 1000
We performed annotation of MACS2 peaks and diffReps regions to genomic features (TSSs, exons, introns, and intergenic regions) using the annotatePeaks.pl module in HOMER (v.3.12)
We mapped RNA paired directional reads to A.gambiae PEST strain genome version 4.3 using TopHat v2.0.13. We aligned reads using the option of library type set as first-strand for directional RNA-seq
Quantification and differential gene expression analysis was conducted using HTSeq/DESeq2 packages
Genome_build: AgamP4.3 (VectorBase)
Supplementary_files_format_and_content: Tracks of ChIP-seq signal, normalized and input corrected, per histone and condition (bw). The bedGraph files were built with MACS2 “bdgcmp" module (-m ppois) and bigwig files using bedGraphToBigWig tool
Supplementary_files_format_and_content: RNA-seq mapped reads coverage was obtained in bigwig format (_coverage.bw) using bamCoverage tool
Supplementary_files_format_and_content: Counts of normalized (RPKM) and input corrected ChIP-seq reads per histone and condition (txt). The counts were obtained using bedtools intersect -c
|
| |
|
| Submission date |
Sep 17, 2018 |
| Last update date |
Jan 08, 2019 |
| Contact name |
Jose Luis Ruiz Rodriguez |
| Organization name |
IPBLN-CSIC
|
| Street address |
Avda. del Conocimiento 17. P. T. Ciencias de la Salud
|
| City |
Granada |
| State/province |
Granada |
| ZIP/Postal code |
18016 |
| Country |
Spain |
| |
|
| Platform ID |
GPL17644 |
| Series (1) |
| GSE120076 |
Chromatin changes induced by a Plasmodium falciparum infection in Anopheles gambiae |
|
| Relations |
| BioSample |
SAMN10079536 |
| SRA |
SRX4705508 |
