We designed 1,143 hairpins targeting 226 lincRNA genes. Of these, we successfully cloned 1,010 hairpins targeting 214 lincRNAs. These hairpins were cloned into a vector containing a puromycin resistance gene and incorporated into a lentiviral vector as previously described22. Briefly, synthetic double stranded oligos that represent a stem-loop hairpin structure were cloned into the second-generation TRC (the RNAi Consortium) lentiviral vector, pLKO.5; the expression of a given hairpin produces a shRNA that targets the gene of interest. Lentivirus was prepared as previously described22. Briefly, 100ng of shRNA plasmid, 100ng of packaging plasmid (psPAX2) and 10ng of envelope plasmid (VSV-G) were used to transfect packaging cells (293T) with TransIT-LT1 (Mirus Bio). Virus was harvested 48 and 70 hours post-transfection. Two harvests were combined. Virus titers were measured as previously described22. Briefly, we measured virus titers by infecting A549 cells with appropriately diluted viruses. 24 hours post infection, puromycin was added to a final concentration of 5ug/ml and the selection proceeded for 48 hours. The number of surviving cells, which is correlated to virus titer, was measured by alamarBlue (BioSource) staining utilizing the Envision 2103 Multilabel plate reader (PerkinElmer)
