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Status |
Public on Nov 17, 2024 |
Title |
Midgut_KUMstrain, MockBlood_Day2, scRNAseq |
Sample type |
SRA |
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Source name |
midgut
|
Organism |
Aedes aegypti |
Characteristics |
tissue: midgut cell type: midgut cells age at_treatment: 5-7 day-old female treatment: blood meal collection time: 2 days after blood meal
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Treatment protocol |
Five- to 7-day-old female mosquitoes were deprived of 10% sucrose solution 18-24 hours before exposure to an artificial blood meal consisting of a 2:1 mix of washed rabbit erythrocytes (BCL) supplemented with 10 mM adenosine triphosphate (Sigma) and Leibovitz’s L-15 medium (Gibco) supplemented with supplemented with 0.1% penicillin/streptomycin (pen/strep; Gibco ThermoFisher Scientific), 2% tryptose phosphate broth (TBP; Gibco Thermo Fischer Scientific), 1× non-essential amino acids (NEAA; Life Technologies) and 10% fetal bovine serum (FBS; Life Technologies). Mosquitoes were exposed to the blood meal for 15 min through a desalted pig-intestine membrane using an artificial feeder (Hemotek Ltd) set at 37°C. Fully engorged females were incubated at 28°C, 70% relative humidity and under a 12-hour light-dark cycle with permanent access to 10% sucrose.
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Growth protocol |
Experiments were conducted with Ae. aegypti mosquitoes derived from a wild-type colony originating from Kumasi, Ghana105. Mosquitoes were reared under controlled conditions (28°C, 12-hour light/12-hour dark cycle and 70% relative humidity). Prior to performing the experiments, their eggs were hatched synchronously in a SpeedVac vacuum device (Thermo Fisher Scientific) for 45 minutes. Their larvae were reared in plastic trays containing 1.5 L of tap water and supplemented with Tetramin (Tetra) fish food at a density of 200 larvae per tray. After emergence, adults were kept in BugDorm-1 insect cages (BugDorm) with permanent access to 10% sucrose solution.
|
Extracted molecule |
total RNA |
Extraction protocol |
A cold dissociation method was used for the mosquito midgut and fat body, adapting protocols originally developed for dissociating Anopheles sp. midgut into single-cell suspensions suitable for scRNAseq (DOI: dx.doi.org/10.17504/protocols.io.j8nlke246l5r/v1, remove the private link prior to publication). Mosquitoes were dissected on ice and pools of 7 organs collected in a drop of ice-cold SF900 III media (Sigma-Aldrich). Each organ was sectioned into 3 smaller pieces using spring scissors (Fine science tools). Sectioned organs were transferred immediately to a chambered coverglass well (Thermo scientific) containing 500ul of dissociation media (SF900 III media supplemented with 10mg/ml Bacillus licheniformis protease (Sigma-Aldrich) and 25 unit/ml DNaseI (Sigma)) for 15 minutes in the dark on ice. Tissues were then gently triturated using 15 back-and-forth pipetting motions. After 5 minutes, 100uL of supernatant containing dissociated cells was collected and transferred to a collection tube containing 45 mL of SF900 III media on ice. The dissociation well was supplemented with 100uL of fresh dissociation media and incubated for 10 min in the dark on ice. Tissues were triturated again, and 100uL of supernatant transferred to the collection tube. Three to four more dissociation rounds were performed until only single cells remained. The collection tube was then spun at 300g for 15 min at 4°C. The pellet was resuspended gently in 900 ul ice-cold SF900 II media and filtered through Flowmi cell strainers (Sigma) with a 40 um porosity for midguts and 70 um porosity for fat bodies. The resulting cell suspension was spun at 300 g for 5 min at 4°C and resuspended in 30 ul of PBS 1x-BSA 0,04%. Library was performed according to manufacter's instruction (Single Cell 5' Kit v2 dual index,10x Genomics).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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|
Description |
10X Genomics
|
Data processing |
STARsolo raw output files were individually read into RStudio (R version 4.3.2 from 2023-10-31) and processed using Seurat (Seurat v5.0.1) and SingleCellExperiment (version 1.24.0). Samples were converted to Seurat objects and SingleCellExperiment objects. A first filter was applied by using the DropletUtils::barcodeRanks function (DroptletUtils version 1.22.0): the knee point of the barcoderank was used as a cutoff to remove empty and /or bad quality droplets with a total UMI number under this value (cf : table with inflection per sample). Assembly: AaegL5 Supplementary files format and content: Tab-separated values files and matrix files
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Submission date |
Nov 12, 2024 |
Last update date |
Nov 17, 2024 |
Contact name |
Thomas Vial |
E-mail(s) |
thomas.vial@pasteur.fr
|
Organization name |
Institut Pasteur
|
Street address |
28 rue du Docteur Roux
|
City |
Paris |
ZIP/Postal code |
75015 |
Country |
France |
|
|
Platform ID |
GPL28657 |
Series (1) |
GSE281673 |
Single-cell atlases of Aedes aegypti midgut and fat body reveal cellular and metabolic dynamics after a blood-meal |
|
Relations |
BioSample |
SAMN44695896 |
SRA |
SRX26693899 |