Arrayed shRNA Screening to Identify Suppressors of Anchorage-Independent Growth

Abstract

Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500–1000 short-hairpin RNAs (shRNA) in “one-shRNA-one-well” format in parallel. These protocols can also be used to conduct functional cDNA or CRISPR screens for modulators of anchorage-independent growth.