title: RNA-seq analysis of Drosophila melanogaster midgut during Pseudomonas entomophila infection
design: RNA-seq analysis of Drosophila melanogaster midgut during Pseudomonas entomophila infection
library_construction_protocol: Pseudomonas entomophila (P.e.) was used for natural (oral) infections. Briefly, for oral infection, flies (app. 7 days of age, except for flies fed oleic acid supplemented food prior to infection) were placed in a fly vial with food/bacteria solution and maintained at 25 °C.  Intact fly (genotype: NP1Gal4>UAS-Lucif.RNAi) midguts were dissected in PBS.  Tissues were collected in 200 µL of Trizol and kept on ice, then homogenized mechanically on ice before adding an additional 800 µL of Trizol to bring the total volume to 1 mL. Samples were incubated on ice for 5 min, followed by the addition of chloroform at one-fifth the total volume (200 µL) with gentle inversion. After a 15 min incubation on ice, samples were centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase (approximately 450 µL) was carefully transferred into a clean tube and mixed with an equal volume of isopropanol (450 µL). Samples were incubated at −80 °C for 30 min and then centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was discarded, and the pellet was washed with 1 mL of ice-cold 75% ethanol by gentle inversion. After centrifugation at 12,000 g for 5 min at 4 °C, the supernatant was removed and the ethanol wash was repeated under the same conditions. The final pellet was allowed to air-dry until nearly translucent and then resuspended in 30 µL of RNase-free water to fully dissolve the RNA. Total RNA was used as template to generate sample libraries for RNA sequencing (Illumina Stranded mRNA Prep Kit). 