Illumina HiSeq 3000 sequencing; RNA-seq of haemoless drosophila larvae compared to matching control animals
RNA-seq of haemoless drosophila larvae compared to matching control animals
P2AIII and attP2 (control) were crossed to uas-rpr (X). Crosses were put in collection cages containing apple juice agar plates at kept at 25oC in a 12h light/dark cycling incubator. 1st instar larvae were picked from apple agar plates and placed into vials containing standard fly at 100 larvae/15mL vial. Larvae were reared at 25oC in a 12h light/dark cycling incubator until further experimentation. 10 male larvae were collected at 3rd instar wandering stage across independent replicates per sample and snap-frozen in liquid N2.  10 male larvae were collected across independent replicates per sample and snap-frozen in liquid N2. Larvae were transferred to Lysing Matrix E homogenization tubes with 1ml of TRIzol and ruptured on high settings inf a FastPrep tissue homogenizer. The supernatant was transferred to a fresh tube and spun down for 2 minutes at max speed. 800l of TRIzol sample was transferred to prespun phaselock tube and 200l Chloroform added. Phases were separated by spinning at 12000 rcf, 15 min, 4C. Upper aqueous phase was transferred to a fresh tube and mixed with 500 l isopropanol. The resulting mix was spun at 20000 rcf, 15 min, 4C. All supernatant was then drained and the pellet resuspended in 30 l DNAse solution (Ambicon, final conc. 0.2U/l) and incubate for 1h at 37C. RNA was purified using the RNeasy Plus kit by addid 270l RTL buffer and then isolating according the the manufacturers instructions.  Libraries were generated by the Max Planck Genome Centre (Cologne). Libraries were stranded, from polyA enriched RNA.