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Sample GSM5532368

Query DataSets for GSM5532368

Status

Public on Jul 22, 2024

Title

Body of flies fed P.aeruginosa (rep B)

Sample type

SRA

Source name

Body of infected house flies

Organism

Musca domestica

Characteristics

tissue: Body
experimental group: Infected

Treatment protocol

One day old adult female house flies were fasted overnight and then individually housed and fed a 2 µl droplet of either P. aeruginosa (4.6 ×105 CFU), E. coli (3.8×105 CFU), or corresponding broth media (controls), as described in Nayduch et al. (2018, J Med Entomol 55:1264-1270).

Growth protocol

P. aeruginosa PAK-gfp and E. coli DH5ɑ were cultured and grown as described in Joyner et al. (2013, PLoS One 8:e79224) and Kumar and Nayduch (2016, Med Vet Entomol 30:218-228).

Extracted molecule

polyA RNA

Extraction protocol

Flies were dissected 4 h post infection to separate the alimentary canal (i.e., gut) from the carcass (remainder of the body). For flies fed E.coli or the corresponding broth media, tissue samples from 3 individual flies were pooled for RNA extraction from each replicate. For flies fed P. aerugionas or the corresponding broth media, RNA was extracted from dissected tissues of individual flies, and RNA from 3 flies was pooled for each replicate. We collected three replicates per tissue (gut or carcass) for the E.coli treatment or corresponding control and four replicates per tissue for the P. aeruginosa treatment or corresponding control. RNA was extracted using Direct-zol miniprep plus kit (Zymo) following the manufacturer’s instructions with an optional TRIzol and BCP phase separation step prior to column purification.
RNA-seq libraries were constructed and sequenced by Novogene. Each RNA sample was normalized to a standard input concentration (1 μg of total RNA) using quantitative PCR, and an Illumina compatible sequencing library was prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer's recommended procedures. The resulting cDNA libraries were size validated on an Agilent 2100 Bioanalyzer System to ensure proper fragment distribution (∼260 bp) and effective removal of adapter dimers. Sequence libraries were multiplexed, and paired-end reads for each sample (2x150bp) were collected on an Illumina NovaSeq 6000 S4 flow cell to an approximate depth of 20 million read pairs for each sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

kallisto v 0.44.0 (Bray et al., 2016) was used to perform pseudo alignment of RNA-seq reads to annotation release 102 of the house fly reference transcriptome from genome assembly v2.0.2 (Scott et al., 2014).
Transcripts were matched to their corresponding genes. Genes with alternative splicing will have multiple transcripts, and we measured the expression of a gene by summing over the count values of all of its transcripts.
Transcript per million (TPM) values were summed for genes with multiple transcripts to obtain gene-level TPM values
Genome_build: Annotation release 102 of the house fly reference transcriptome from genome assembly v2.0.2 (GCA_000371365.1)
Supplementary_files_format_and_content: Pseudomonas_infected_counts.csv
Supplementary_files_format_and_content: Ecoli_infected_counts.csv
Supplementary_files_format_and_content: Pseudomonas_infected_TPM.csv
Supplementary_files_format_and_content: Ecoli_infected_TPM.csv

Submission date

Aug 23, 2021

Last update date

Jul 22, 2024

Contact name

Richard Meisel

Organization name

University of Houston

Department

Biology and Biochemistry

Street address

3455 Cullen Blvd