Materials and Reagents

1. Human lung adenocarcinoma cell line PC9 (RIKEN BioResource Center, catalog number: RCB4455 )
   
2. RPMI 1640 Medium containing L-Glutamine (Life Technologies, Gibco^®, catalog number: A10491-01 )
   Note: For cell culture, the medium is supplemented with 10% FBS and 1% Penicillin Streptomycin (see details FBS and Pen/Strep below).
   
3. Penicillin streptomycin (Life Technologies, Gibco^®, catalog number: 15070-063 )
   
4. Fetal bovine serum (FBS) (PAA Laboratories GmbH, catalog number: A15-101 )
   
5. Opti-MEM^® I Reduced Serum Medium (Life Technologies, Gibco^®, catalog number: 31985-047 )
   
6. 384-well tissue culture plates, black with clear F-bottom (4titude, Anachem, catalog number: 4TI-0201 were used for our screen; currently plates from Greiner bio-one (catalog number: 781091 ) are used
   
7. Adhesive foil plate seal (Brandle Plate Sealer, Alpha Biotech)
   
8. 5x siRNA buffer (GE Healthcare, catalog number: B-002000-UB-100 )
   
9. Human siGENOME SMARTpool siRNA library (GE Healthcare, catalog number: G-005005 )
   Note: We used an older version (from 2005) for our study (de Bruin et al., 2014) that is no longer available. Updated versions will contain other siRNA sequences as well as annotations, which will impact results. We therefore strongly recommend validating identified genes using other approaches.
   
10. siRNA controls (see Note 1)
    Negative controls:Human siGENOME non-targeting siRNA 2 (GE Healthcare, catalog number: D-001210-02 ), RISC-Free control siRNA (catalog number: D-001220-01 ), ON-Targetplus non-targeting (catalog number: D-001810-10 )
    Positive controls: Human siGENOME SMARTpool UBB (GE Healthcare, catalog number: M-013382-01 ) and PLK1 (Dharmacon, catalog number: M-003290-01 )
    
11. DharmaFECT 2 siRNA transfection reagent (GE Healthcare, catalog number: T-2002-01 ) (see Note 2)
    
12. DAPI (Roche Diagnostics, catalog number: 10236276001 )
    
13. siGENOME LaminA/C control siRNA (GE Healthcare, catalog number: D-001050-01-05 ) (see Note 3)
    
14. Human LaminA/C antibody (catalog number: SC-7292 ) (see Note 3)
    
15. Phosphate buffered saline (PBS) (pH 7.0, made in-house)
    
16. Erlotinib (Enzo Life Sciences, catalog number: BML-DL279 )
    

Equipment

1. Acumen (TTP LABTECH, mode: eX3 )
   
2. ArrayscanVTi HCS microscope (Cellomics)
   
3. Biomek FX Liquid handling platform (Beckman Coulter)
   
4. Countess Cell counter (Life Technologies)
   
5. Liquid dispensor8-channel [for our screen the WellMate (Matrix) was used, currently the FluidX (Xrd-384) is used]
   
6. Cytomat 24 °C CO₂ incubator 37 °C and 5% CO₂
   Note: The Cytomat is a cell culture incubator that has racks built in holding ~500 plates, and the racks rotate to minimize plate edge effects.
   
7. Plate washer (BioTek Instruments, model: ELx405 Select CW , Figure 1)
   
8. Plate sealer (Brandel Plate Sealer, Alpha Biotech)
   
   
   Figure 1. Picture of plate washer
   

Important considerations

Before performing a large-scale siRNA screen, there are a few crucial points to consider and to optimize in order to obtain the best results (Jiang et al., 2011). These include:

1. Optimization of the siRNA transfection: Reagent, cell number, siRNA concentration.
   
2. Optimization of the drug dose, treatment time length in combination with the siRNA transfection for drug resistant/sensitizing screens.
   

Details of these optimization steps are provided in the Notes section at the end of this protocol. We recommend performing, if possible, a pilot screen with a range of random siRNAs and selected control RNAs to determine the feasibility and screen read-out prior to the genome-wide screen.

Procedure