HCA2 or IMR-90 (early passage cells)
Lentivirus (containing shRNA – GFP Ctrl and target gene)
Growth medium: DMEM, 10% FBS, 1% antibiotic – warm to 37°C
Freezing medium: 90% growth medium + 10% DMSO
Trypsin – warm to 37°C
1X PBS (without calcium) – warm to 37°C
15 ml tubes, 10% bleach

1. Cell Plating
   Seed HCA2 at 1 x 105 cells/well in 6 well plates containing 2 ml growth medium Incubate at 3% O₂, 10% CO₂ to ~40% confluent (1day)

2. Infection [Make sure to disinfect with bleach ALL materials that came into contact with virus/virus media]
   In 1.5 ml tubes, prepare the following:
   3 µl of 4 mg/ml polybrene (final con’c = 6 µg/ml)
   2X, 0.4X and 0.08X of virus suspension
   960 µl of growth medium
   Aspirate media from cells then add virus mix (save 2 no virus wells) and incubate overnight at 3% O₂, 10% CO₂

3. Change media
   24hrs latter, treat cells 1 µg/ml Puromycin (save 1 control non treated).
   Release from puro after 3 days. Select the virus concentration that gave the highest NON 100% infection rate (based on cells survival to puro). Scale up!