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Sample GSM8625521 Query DataSets for GSM8625521
Status Public on Nov 17, 2024
Title Midgut_KUMstrain, MockBlood_Day2, scRNAseq
Sample type SRA
 
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
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.
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).
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
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
 
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

Supplementary file Size Download File type/resource
GSM8625521_TV_M2_filtered_barcodes.tsv.gz 55.1 Kb (ftp)(http) TSV
GSM8625521_TV_M2_filtered_features.tsv.gz 115.0 Kb (ftp)(http) TSV
GSM8625521_TV_M2_filtered_matrix.mtx.gz 22.0 Mb (ftp)(http) MTX
GSM8625521_TV_M2_raw_barcodes.tsv.gz 2.1 Mb (ftp)(http) TSV
GSM8625521_TV_M2_raw_features.tsv.gz 115.0 Kb (ftp)(http) TSV
GSM8625521_TV_M2_raw_matrix.mtx.gz 51.2 Mb (ftp)(http) MTX
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Raw data are available in SRA

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