title: GF-d4-Plate2-28208-93
organism: Drosophila melanogaster
description: Protocols: The 53 DGRP lines were obtained from Bloomington Drosophila Stock Center. Drosophila were kept at 25°C in a Panasonic Mir425 incubator with 12/12 hrs dark/light cycles. Routine stocks were kept on standard laboratory diet. Field-collected flies were trapped with rotten tomatoes in a garden in Solaize (France) and reared on a medium without chemical preservatives to minimize the modification to their gut microbiota. To generate DGRP F2, four DGRP lines were selected for setting up seven different crosses. All RNAi lines were crossed to the driver lines. To minimize lethality, we dampened the GAL4 strength by leaving the genetic crosses at 25°C. All live Drosophila larvae were collected from each nutritive cap containing low yeast diet by temporary immersion in sterile PBS, killed with a short pulse of heat treatment (5 sec at 90°C), transferred on a microscopy slide, mounted 80% glycerol/PBS. For each DGRP strain and each cross and/or condition, at least three biological replicates were generated. For each mono-association experiment, Lp WJL was grown in Man, Rogosa and Sharpe (MRS) medium over-night at 37°C, and diluted to O.D.=0.5 the next morning to inoculate 40 freshly laid eggs on each 55mm petri dish or standard 28mm tubes containing fly food of low yeast content. The inoculum corresponds to about 5x10 7 CFUs. Equal volume of sterile PBS was spread on control axenic eggs. To contaminate the garden-collected flies with their own microbiota, eggs were dechorionated and directly seeded onto appropriate food caps. Sterile PBS was used to wash the side of the bottles where the adult wild flies were raised to recover more fecal content, and 300 ul of the wash was inoculated to the dechorionated eggs. For GF control, 300 ul of sterile PBS was used to inoculate the dechorionated eggs. Total RNA was isolated using TRIzol (Sigma) and followed by double precipitation with ethanol. BRB-seq (see https://www.biorxiv.org/content/early/2018/01/30/256594)