Plate Format:

384 well

Origin:	Tapon Lab
Cell Medium:	Schneiders Drosophila media + 10% heat-inactivated serum + P/S
Cell Seeding Density Per Well:	6000

Protocol:	Thaw the ds RNA library plates for 30mins at RT, then spin. Dilute the control dsRNA into 220ng per 5ul in HBSS and then add 5ul per well into the control wells. The amount of plasmids Yki-NTEV, 14.3.3eps-CTEV, GV-2ER (each 10ng/well), UAS-Fluc (20ng/well) and pIZ-Rluc (3ng/well) added per well. Mix the DNA with FuGENE HD, incubate at RT for 10min. Then mix with S2R+ cells, then loading the cells into the plates. FuGENE HD is 0.25ul per well. And the cells is 6000cells/well. The total transfection volume is 75ul per well.
Delivery Reagent Concentration:	0.25ul/well
Silencing Reagent Concentration:	220ng dsRNA per well
Silencing Reagent Supplier:	Ambion
Silencing Reagent Origin:	Tapon Lab

Cell lysis
Treatment:Aspirate the culture media, add 5ul/well 1x passive lysis buffer
Compounds:passive lysis buffer(1x)
Compound Concentration:5ul/well
Treatment Time:15min
Firefly luciferase activity
Treatment:Add luciferase assay reagent II 5ul/well to read
Compounds:luciferase assay reagent II
Compound Concentration:5ul/well
Treatment Time:Read immediately
Renilla luciferase activity
Treatment:Add Stop&Glo reagent 5ul/well to read
Compounds:Stop & Glo reagent
Compound Concentration:5ul/well
Treatment Time:Read immediately