Genome-wide shRNA screen for fluvastatin targets

A549 cell line, stably transduced with the RNAi Consortium (TRC1) shRNA library [27 (link)] was treated with fluvastatin (US Biologicals) over 12 days. Genomic DNA (gDNA) was amplified and shRNA populations hybridized onto custom Affymetrix Gene Modulation Array Platform (GMAP) arrays. Differences between shRNA abundances over 12 days were assessed using the Bayes Factor [29 (link)]. For generation of stably expressing shRNA cell lines, DNA from bacterial glycerol stocks of shRNAs from the TRC1 library was amplified using E.coli and purified (Qiagen Plasmid Maxiprep kit). Lentiviral particles were generated by calcium phosphate transfection of sub-confluent (50–60%) HEK293TV cells with 10 μg of TRC pLKO.1 puro shRNA construct, 5 μg each of pMDG1.vsvg, pRSV-Rev and pMDLg/pRRE constructs. Lentiviral particles were collected 24 and 48 hours later, filtered though a 0.45 μm filter and stored at −80°C. Parental cell lines were infected with lentiviral particles containing the indicated shRNAs and puromycin selected (48 hrs, 2 μg/ml of puromycin).