Genome-wide CRISPR screen for export factors of NORAD lncRNA

Fluorescence based genome-wide screening strategy in which the endogenous NORAD transcript was tagged with an IRES-GFP cassette that could only be translated after successful transport to the cytoplasm. Infection of these cells with a genome-wide lentiviral CRISPR library followed by the identification of sgRNAs that were enriched in cells with diminished fluorescence Overall design: Brunello lentiCRISPR_v2 library (Addgene #73179) was used. The two independent reporter cell lines served as biological replicates. In each replicate, 19 million cells were seeded into seven 15 cm dishes each, in media containing 8 µg/mL of polybrene (Millipore) and lentiviral library at a multiplicity of infection of ~0.5. Two days post-transduction, cells were seeded into media containing 1 µg/mL puromycin (Thermo Fisher Scientific). Cells were selected in puromycin containing media for the next ten days, during which they were passaged every two days. On day 10 in puromycin, the dimmest 0.5% cells were sorted to collect at least 200 000 cells per replicate in addition to 50 million unsorted cells per replicate.