GSM9821597: Mature Replciate 4; Drosophila melanogaster; RNA-Seq
Brains were dissected in cold Schneider's media. Brains were first enzymatically dissociated in Papain (100U/ml) suspended in 1X PBS and supplemented with 0.18U/ml LiberaseTM cocktail (Roche). Dissociation was carried out for 50 min at room temperature with nutating. Dissociated brains were rinsed three times in cold Schneider's to stop dissociation. Schneider's medium was replaced by 2% Bovine Serum Albumin in 1XPBS.  Mechanical trituration was performed by first pipetting the brains up and down 100% in P200 pipette tips pre-coated with fetal bovine serum; then, dissociated tissue was passed 20 times through a 3cc gauge needle pre-coated with fetal bovine serum. The suspension was then transferred to FACS tubes kept on ice. DAPI was added to label dead cells.  In parallel, +>TdTomato brains were treated in parallel to gate TdTomato signal during FACS sorting. Sorting was performed using an Aria cytometer equipped with a  450 nm UV and 582 nm lasers and a 100 um nozzle. RNA was prepared by Admera using the SMARTseq V4 with Nextera XT