GSM5532372: Gut of flies fed P.aeruginosa (rep B); Musca domestica; RNA-Seq
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.