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Sample GSM5808262

Query DataSets for GSM5808262

Status

Public on Mar 01, 2022

Title

Ae. aegypti ovary 72 hours/3 days post-blood-meal with eggs retained replicate 2

Sample type

SRA

Source name

Ae. aegypti Ovary

Organism

Aedes aegypti

Characteristics

strain: Liverpool
tissue: Ovary
group: 72 hours/3 days post-blood-meal with eggs retained
age: 19-to-20-day old adults
genotype: Wild type
Sex: Female

Extracted molecule

total RNA

Extraction protocol

RNA extraction was done using the PicoPure Kit (ThermoFisher, KIT0204), with one modification to homogenize tissue: instead of lysis buffer, 100 µL of TRIzol (ThermoFisher, 15596026) was added to the collection tube on ice. Manual tissue homogenization was done for 30 seconds using the Pellet Pestle Motor (Kimble, 749540) and RNase-Free pellet pestle (VWR, KT749510-0590) with 140 µL of TRIzol added to a total volume of 240 µL. After 5 minutes of room temperature incubation, 48 µL of chloroform:isoamyl alcohol 24:1 was added (Sigma, C0549), and tubes were shaken by hand for 30 seconds and left to stand for 2 minutes. The aqueous TRIzol layer was removed after centrifuging (12,000 RPM for 15 minutes at 4°C) and added to the PicoPure column, <130 µL at a time. All subsequent steps, including DNAse treatment, were performed following PicoPure manufacturer protocols.
RNA-seq libraries were prepared using Illumina TruSeq stranded mRNA LT kit (Illumina, 20020594) following manufacturer protocol using 100 ng of total RNA. Libraries prepared with unique dual indexes were pooled at equal molar ratios.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

72hours_postbloodmeal

Data processing

Sequence and transcript coordinates for the Aedes aegypti mosquito genome and gene models were obtained by merging the Aaeg_L5 RefSeq annotation from NCBI with a manual chemoreceptor annotation. Information related to generating this annotation is available at https://github.com/VosshallLab/Jove_Vosshall_2020/tree/master/RNAseq_merged_annotation from Jové et al., 2020a (PMID: 33049200)
Transcript expression was calculated using the Salmon quantification software (version 0.8.2) from Patro et al., 2017 (PMID: 28263959). Gene expression levels were obtained using Tximport (version 1.8.0) as transcripts per million (TPMs).
For sample variability assessment (and any differential expression analysis), raw read counts in genes were normalized and rlog transformed using DESeq2 (version 1.20.0) as per Love et al., 2014 (PMID: 25516281). Subsequently, principal component analysis was performed, and sample distance correlations were calculated to assess between sample variability with hierarchical clustering.
Genome_build: aal5_genome.fa
Supplementary_files_format_and_content: tab-delimited text file of abundance measurements in transcripts per million (TPM) for all samples

Submission date

Jan 11, 2022

Last update date

Mar 01, 2022

Contact name

Krithika Venkataraman

Organization name

The Rockefeller University

Lab

Laboratory of Neurogenetics and Behavior

Street address

1230 York Ave