Table 1.
Assay Workflow Using the Optimized Conditions and Experimental Steps for the Execution of the Genome-Wide shRNA Lethality Screen in HeLa Cells
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Cell plating | 45 μL | 500 cells per well in growth media containing 8 μg/mL polybrene |
| 2 | Incubation time | 24 h | 37°C, 5% CO2, dedicated Cytomat incubator, controlled humidity |
| 3 | Cell transduction | 4 μL | Arrayed lentiviral particle shRNA library |
| 4 | Assay plate spin | 8 min | Spin assay plate on bench top centrifuge at 340 g |
| 5 | Incubation time | 48 h | 37°C, 5% CO2, dedicated Cytomat incubator, controlled humidity |
| 6 | Puromycin selection | 50 μL | Growth media containing 3 μg/mL puromycin |
| 7 | Incubation time | 96 h | 37°C, 5% CO2, dedicated Cytomat incubator, controlled humidity |
| 8 | Cell recovery | 50 μL | Growth media |
| 9 | Incubation time | 24 h | 37°C, 5% CO2, dedicated Cytomat incubator, controlled humidity |
| 10 | Fix | 50 μL | Wash twice with 1×PBS, 4% paraformaldehyde (v/v) for 20 min |
| 11 | Nuclei staining | 50 μL | 10 μM Hoechst solution containing 0.05% Triton X-100 (v/v) |
| 12 | Image acquisition | 360 nm/450 nm (ex/em) | INCA2000 automated microscope |
| 13 | Image analysis | Multiparametric analysis using Developer Toolbox 1.7 software |
Step Notes
1. Dispense cells into assay plates with Multidrop 384.
3. Transfer lentiviral particles on the PP-384-M Personal Pipettor using a custom 384 head; 30 s per plate.
6,8,10. Aspirate on the ELx405 automated washer and dispense with Multidrop 384; 1 min per plate.
11. Dispense into assay plates with Multidrop 384.
12. 4 s per well with a total imaging time of 25.6 min per plate.
13. Analysis of Hoechst-stained nuclei, 8 min per plate.