18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences
Cell Culture and Flavopiridol Treatment Formost RAP experiments, we used V6.5malemouse ES cells grown in 2i + LIF on plates precoated with 0.2%gelatin and 3.5 mg/mL laminin. For Xist RAP-DNA, we used pSM33malemouse ES cells inducedwith doxycycline for three hours to activate Xist expression as previously described (Engreitz et al., 2013). For Xist RAP-RNA, we used pSM33s inducedwith doxycycline for six hours to increase total Xist abundance and thus provide a control with expression better matched to that of Malat1. For transcription inhibition, we treated pSM33s (in the absence of doxycycline) for one hour with 1 mM flavopiridol or vehicle only (DMSO). We first made a 1 mM flavopiridol stock solution by resuspending flavopiridol hydrochloride hydrate (Sigma) in DMSO, and then added flavopiridol to 1 mM in cell culture media. We harvested cells after one hour to minimize effects due to flavopiridol toxicity. We note that replicate RAP experiments across the two male mouse ES cell lines (V6.5 and pSM33) produced similar data.