We cloned 5 shRNAs targeting each lincRNA into a puromycin-resistant lentiviral vector22. ESCs were plated on pre-gelatinized 96-well plates and infected with lentivirus prior to addition of irradiated DR4 MEFs. Media containing 1ug/mL puromycin was added 24 hr after infection. On-target knockdown was assessed after 4 days and the best hairpin showing a knockdown >60% was selected. RNA from 147 lincRNAs, 40 protein-coding genes, and 27 negative controls were hybridized to Agilent microarrays. Differentially expressed genes were defined as having an FDR<5% and fold-change >2-fold compared to controls.
