To perform loss-of-function experiments, we generated five lentiviral-based short hairpin RNAs (shRNAs)22 targeting each of the 226 lincRNAs previously identified in ESCs2,3 (see Methods, Supplemental Table 1). These shRNAs successfully targeted 147 lincRNAs and reduced their expression by an average of ~75% compared to endogenous levels in ESCs (see Methods, Figure 1a, Supplemental Figure 1, Supplemental Table 2). As positive controls, we generated shRNAs targeting ~50 genes encoding regulatory proteins, including both transcription and chromatin factors that have been shown to play critical roles in ESC regulation17,20,23; validated hairpins were obtained against 40 of these genes (Supplemental Table 2). As negative controls, we performed independent infections with lentiviruses containing 27 different shRNAs with no known cellular target RNA.
