To explore how lincRNAs carry out their regulatory roles, we studied whether lincRNAs physically associate with chromatin regulatory proteins in ESCs. We previously showed that many human lincRNAs can interact with the polycomb repressive complex4, a complex that plays a critical functional role in the regulation of ESCs18,19. To determine whether the ESC lincRNAs physically associate with the polycomb complex, we crosslinked RNA-protein complexes using formaldehyde, immunoprecipitated the complex using antibodies specific to both the Suz12 and Ezh2 components of Polycomb, and profiled the co-precipitated lincRNAs using a direct RNA quantification method41 (see Methods). We performed immunoprecipitation of the Polycomb complex across 5 biological replicates and 8 mock-IgG controls, and we assessed significance using a permutation test (see Methods, Supplemental Figure 16). Altogether, we identified 24 lincRNAs (~10% of the ESC lincRNAs) that were strongly enriched for both Polycomb components (Figure 5b, Supplemental Table 17).
