Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms

Complementation assays and immunofluorescence 1-2 million Kc167 cells in 2 ml complete Schneider’s Drosophila Medium were seeded in each well of a 6-well plate. After 4 h, cells were transfected with 500 ng of complementation plasmid (described before) + 25 ng of pCoBlast (Thermo Fischer, Cat. No K5150-01) using Effectene Transfection Reagent (QIAgen, Cat. No 301425). 48 h after transfection, 2 ml of the cells were collected, transferred into T-25 flasks and diluted with 4 ml of complete Schneider’s Drosophila Medium + Blasticidin at a final concentration of 50 ng/ul. 7-8 days after selection the cells were collected and treated with dsRNA as described before. For immunofluorescence, 0.2-0.4 million cells in 200 µL of complete Schneider’s Drosophila Medium were seeded onto a round 12 mm coverslips (Paul Marienfeld GmbH & Co., Cat No. 0117520) placed separately inside wells of 12-well plates. Cell were allowed to attach for 2-4 h and the coverslips were gently rinsed with 500 µL of PBS. Cells were fixed in 500 µL of ice-cold PBS + 2% formaldehyde for 7.5 min. After removal of fixative, cells were permeabilized by adding 500 µL of ice-cold PBS + 0.25% Triton-X-100 + 1% formaldehyde and incubating for 7.5 min. Coverslips were washed two times with 1 ml of PBS and blocked with PBS + 3% BSA for 1h at room temperature. Coverslip were transferred onto a piece of parafilm, placed into a wet chamber and 40 µL of primary antibody solution was gently added onto the coverslip. After overnight incubation at 4°C, coverslips were transferred back to 12-well plates and washed twice with 1 ml of PBS. Coverslip were transferred again onto a piece of parafilm, placed into a wet chamber and 40 µL of secondary antibody was gently added onto the coverslip. After 1 h incubation at room temperature, coverslips were transferred back to 12-well plates and washed twice with 1 ml of PBS. Cells were incubated with 1ml of 0.2 µg/ml DAPI (Sigma-Aldrich, Cat. No 10236276001) for 5 min at room temperature. Coverslips were washed with PBS and with deionized water, mounted on slides with 8 µL of Vectashield (Vector Laboratories, Cat. No H-1000) and sealed with nail polish. Images were taken on a Leica SP5 confocal microscope. Images were processed and analyzed using Fiji. Script available as source code file.