To further validate the role of these 26 lincRNAs in regulating the pluripotent state, we knocked down these lincRNAs in wild-type ESCs and measured mRNA levels of pluripotency marker genes Oct4, Sox2, Nanog, Klf4, and Zfp42 after 8 days. In all cases we observe a significant reduction in the expression of multiple pluripotency markers with >90% showing a significant decrease in both Oct4 and Nanog levels (Supplemental Figure 5, Supplemental Tables 8,9). To control for ‘off-target’ effects, we studied additional hairpins targeting these lincRNAs. For 15 lincRNAs we had an effective second hairpin. In all 15 cases, the second hairpin produced comparable reductions in Oct4 expression levels, showing that the observations were not due to ‘off-target’ effects (Figure 2b, Supplemental Table 10). Notably, >90% of lincRNA knockdowns affecting Nanog reporter levels led to loss of ESC morphology (Figure 2c, Supplemental Figures 6, 7). Thus, inhibition of these 26 lincRNAs lead to an increased exit from the pluripotent state.
