For 137 of the 147 lincRNAs (93%), knockdown caused a significant impact on gene expression (Supplemental Table 3), with an average of 175 protein-coding transcripts affected (range: 20–936) (Figure 1c, Supplemental Figure 2, Supplemental Table 4). These results were similar to those obtained upon knockdown of the 40 well-studied ESC regulatory proteins: 38 (95%) showed significant effects on gene expression, with an average of 207 genes affected (range: 28 (for DNMT3L) to 1187 (for Oct4)) (Figure 1c, Supplemental Figure 2, Supplemental Table 4). Although some individual lincRNAs have been found to lead primarily to gene repression12,13, we find that knockdown of the lincRNAs studied here largely led to comparable numbers of activated and repressed genes (Supplemental Figure 2, Supplemental Table 4). To further assess ‘off-target’ effects, we also profiled the effects of the second-best validated shRNA targeting 10 randomly selected lincRNA genes. In all cases, second shRNAs against the same target produced significantly similar expression changes (see Methods, Supplemental Table 5). These results indicate that the vast majority of lincRNAs have functional consequences on overall gene expression of comparable magnitude (in terms of number of affected genes and impact on levels) to the known transcriptional regulators in ESCs.
