1. Transfection Optimisation Process Each new siRNA screen entering the TDI requires a full transfection optimisation process. For RNAi screens, establishing the optimal transfection conditions is the most important step of the optimization/validation process and this experiment is designed to optimise the screening conditions on the liquid handling robots before entering into pilot or full scale screening. This allows us to: –Select the most appropriate transfection reagent and concentration for the desired phenotypic change while minimizing transfection reagent toxicity. –Select the most appropriate siRNA’s concentrations for maximum knock-down while reducing off-target effects. –Determine accuracy of liquid handling capabilities. –Optimize the transfection protocol by mimicking the conditions we plan to employ producing your high throughput screen Each transfection optimisation plate is able to titre 2 transfection reagents at 4 concentrations (0.2, 0.1, 0.075 and 0.05ul/well)* and 2 siRNA’s at 3 concentrations (20nM, 10nM and 5nM final concentration). The layout produces a matrix of transfection conditions from which we will chose the best result. The conditions and well layouts described below provide sufficient samples for accurate Western Blotting and will be run in triplicate (to mimic the screen production output). * HyperFect requires 7X the amount of siRNA compared to other major vendors.

2. Transfection Optimisation Requirements On the day of the experiment, you must provide adequate volumes of: –Two siRNA’s (min. 1ml of each) in sterile RNAse Free Water. These will be transferred to, 2ml eppendorfs for Varispan picking. These should be siRNA’s you have used in your laboratory and have good antibodies for running Westerns. –Two Transfection reagents (min. 6500ul of each) in sterile Optimem. This will be transferred to 2ml eppendorfs for the Varispan. –Cells (min. 200ml) at the required density in sterile antibiotic free media which will usually be 6000 cells / well. These must be certified mycoplasma free prior to screening. –Antibiotic Free Media (min. 300ml), sterile. –Optimem (min. 100ml) sterile. Janus Protocols to be used: –Varispan - Trans Optim – FINAL.MPT –MDT - Transfection Reagent Addition – FINAL.MPT 800nM siRNA Total Volume20uM siRNA (ul)Optimem vol siRNA 1100040960 siRNA 2100040960 nT50020480 Death Control50020480 Transfection Reagent Total Volume T.Reagent (ul)Optimem vol Transfection Reagent #1 65001306370 Transfection Reagent #1 65001306370

3. 800nM siRNA 1 siRNA 2 200nM 400nM NT DC siRNA Master Plate Varispan Liquid Handler picks siRNA’s into 96 well master plate from 2ml Eppendorf tubes and makes 1:2 serial dilutions in 96 well plate. SiRNA 1 top conc added to column 1A-D siRNA 2 top conc added to column 1 E-H Non Target added to column 4, Death Control to column 5 Serial dilution of siRNA 1 and 2 across columns 2 and 3. 200nM400nM800nM siRNA 1 siRNA 2 siRNA working stock plate Varispan Liquid Handler picks siRNA’s into 96 well master plate from siRNA Master plate siRNA 1 added at 800, 400 and 200nm siRNA 2 added at 800, 400 and 200nm 30ul/ well NT and Death Control working stock Plate Varispan Liquid Handler picks siRNA’s into 96 well master plate from siRNA Master plate 30ul/ well Non TargetDeath siRNA working stock plates Varispan Liquid Handler picks transfection reagents into working stock plates and performs a 1:2 titration down plate. Transfection Reagent #1 0.5ul/well 0.075ul/well 0.1ul/well 0.2ul/well 0.5ul/well 0.075ul/well 0.1ul/well 0.2ul/well Transfection Reagent #2 Transfection Reagent #1 200nM400nM800nM siRNA 1 siRNA 2 200nM400nM800nM siRNA 1 siRNA 2 Transfection Reagent #1 Transfection Reagent #2 Non TargetDeath siRNA complex plates MDT Liquid Handler adds 10ul siRNA to transfection reagent plates creating a 1:2 dilution. 20 minutes complex time followed by addition of 80ul (cells + media) to plates. Transfection Reagent #2 0.5ul/well 0.075ul/well 0.1ul/well 0.2ul/well 0.5ul/well 0.075ul/well 0.1ul/well 0.2ul/well Transfection Reagents Combined Cells Alone

4. Transfection Optimisation Processing Steps 1.Make working stock siRNA plate(s) by making 1000uL of 800nM stock siRNA in 2ml eppendorf tubes 2.Make working stock NT Control and Death Control siRNA’s by making 500uL of 800nM stock siRNA in eppendorf tube: Using Janus, transfer 200uL of siRNA1 and siRNA2 to appropriate wells. Transfer 50uL to 8 wells in column 4 for Non Target (NT) and 50uL to 8 wells in column 5 for Death Control (DC) in 96well costar v-bottom plate. 3.Fill column 2 and 3 with 100uL of Optimem in all wells. 4.Use Janus to make 1:2 serial dilutions of siRNA’s by transferring 100uL across columns 1,2 and 3. 5.Fill 3 plates with 30uL Optimem all wells of siRNA Complexes Plate. 6.Transfer 10uL of siRNAs from siRNA Titration Plate to 2 siRNA Complexes Plate. siRNA 1 siRNA 2 NT DC NT siRNA 1 siRNA 2 DC 800 400200 200 uM100 uM50 uM200 uM100 uM50 uM 200 uM

5. Transfection Optimisation Processing Steps 7.Make Transfection Reagent Plate: Janus will flip Transfection Plates by 90 o V arispan liquid handler will fill Rows A,C,E and G with 100uL of Optimem; then fill row B and F with 62.5uL 8. Make up Transfection reagents (6500ml), aliquot into 2ml eppendorfs Varispan will then transfer 250uL into all wells of Row D and H. Varispan will perform a serial dilution, transferring 37.5ul from Row D to Row B; then 100ul from D to C; then 100ul from C to A. Serial dilution, on second half of plate; transferring 37.5ul from Row H to Row F; then 100ul from H to G; then 100ul from G to E 9.Janus will flip transfection Plates back 90 o. 10.Janus MDT will finish making siRNA Complexes Plate by aliquoting 40ul of transfection reagent from Transfection Reagent Plate to siRNA Complexes Plate. Mix thoroughly and let incubate for 20 minutes. 11.Remove the bottom 4 rows of tips from Box 3 and the top 4 rows from Box4. 12.Use Janus to make Transfection Reagent Control Plate by aliquoting 40ul of transfection reagent from Transfection Reagent Plate to siRNA Complexes Plate nonTargeting. Mix thoroughly and let incubate for 20 minutes. Do the same for the nT Control Plate with Dharmafect 1 to the top 4 rows and RNAiMax to the bottom 4 rows. 13.Fill 4 plates with 30uL Media, add 20uL of siRNA complexes and fill with 50uL of Cells using established Janus protocols. 14.Change the media at 24 hours and grow cells for and additional three days 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well Transfection Reagent #1 Transfection Reagent #2 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well Transfection Reagent #1 Transfection Reagent #2 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well 0.05 uL/well 0.075 uL/well 0.1 uL/well 0.2 uL/well

6. Transfection Optimisation Readout 1.Knock-down will be visually confirmed by cell Viability in the Death Control at the end of day three. 2.On day four (96 hours) the cell viability of all plates will be assessed using resazurin fluorescence readout of cell viability or Hoecht staining to count cells to produce a transfection toxicity curve. See table right for a representative table for transfection reagent toxicity. *Note, 0.2uL TR / well was excessively toxic for this cell line. 2.Westerns will be performed on the all three siRNA concentrations, nT siRNA and Cells alone for each siRNA where the transfection reagent is efficient but not excessively toxic. In total, four NuPAGE® Novex 4-12% Bis-Tris Gel 1.5 mm, 15 well will be run (each siRNA in two transfection reagents for a total of four gels) to identify the optimal siRNA and transfection reagent concentration. *Note the Western for GAPDH using RNAiMax suggest the siRNA transfection concentration was sufficient at 20nM, 10nM and 5nM and 0.075 uL RNAiMax and 0.05 uL RNAiMax / well were not sufficient for a thorough knock-down. The transfection conditions for this screen were optimized at 10nM siRNA concentration and 0.1uL / well RNAiMax.