Copas select

Drosophila stocks were maintained at 25°C on standard cornmeal agar. piggyBac mobilisations were performed as exemplified in the crossing schemes described in supplementary Materials and Methods using J10 or J6 pMos{3×P3-ECFP, αtub-piggyBacK10} transposase sources (Horn et al., 2003 (link)). Virgin collection was simplified by using P{hs-hid}Y to eliminate males (FlyBase). Embryos from dysgenic crosses were collected on freshly yeasted apple-juice agar plates and individual YFP-positive embryos were selected using the COPAS Select (Union Biometrica). Single embryos were collected in 24-well apple juice agar plates, surviving L3 larvae were transferred to individual yeasted cornmeal agar tubes and eclosing adults were crossed as described in supplementary Materials and Methods. Embryos were collected from established lines and YFP expression confirmed by sorting with the COPAS Select. Positive lines were mapped to the Drosophila genome via inverse-PCR or 5′ and 3′ RACE (Liao et al., 2000 (link)). Manipulation of gene lists and assessment of gene ontology enrichments (Holm-Bonferroni corrected for multiple testing and corrected for gene length) were performed in FlyMine (Lyne et al., 2007 (link)).

First, 5 μl of any of the chemical compounds was added to flat-bottomed 96-well plates (Daslab, Barcelona, Spain) and mixed with 200 μl of standard fly food by using a Biomek NXP liquid-handling robot station (Beckman Coulter, Brea, CA). Secondly, L1 larvae [yw; UAS-INSR:Luc#6/+; MHC-Gal4/UAS-i(CTG)₄₈₀] were plated in each well using a mid-size flow cell cytometer (COPAS select, Union Biometrica, Holliston, MA). Finally, each plate was sealed by placing a micro-perforated 96-well plate with deep wells (ABgene, Waltham, MA) in an upside-down position with a punched foam placed between both plates to avoid larvae from passing from one well to another. Six replicates were assayed from each screening plate. All plates, including those later used for the luciferase read-out (see below), were labeled with barcodes for correct sample identification throughout the entire screening process. Flies were allowed to develop in a compound containing food at 25°C for 14 days. At this point, flies reached the adult stage and were frozen at −20°C, prior to quantifying their minigene luciferase levels.