Extended Data Figure 3: Immunoprecipitation of the identified Xist-interacting proteins confirms Xist RNA interaction.

RNA immunoprecipitation experiments were performed for seven Xist-interacting proteins (black bars), two control RNA binding proteins that were not identified by RAP-MS and IgG (grey bars) in UV-crosslinked cell lysate after 6 h of Xist induction by doxycycline addition (Methods). The RNA associated with each protein was measured and enrichment levels were computed relative to the level of the RNA in total cellular input and normalized to the total efficiency of capture in each sample to allow for direct comparison across all immunoprecipitation experiments (Methods). a, Enrichment of the Xist lncRNA after immunoprecipitation from a sample of pSM33 male cells. b, Immunoprecipitation of SHARP was performed from a sample of UV-crosslinked females ES cells that were treated with retinoic acid for 24 h. The levels of recovered Xist lncRNA (black bars), Neat1 lncRNA (white bars), and 45S pre-ribosomal RNA (grey bars) were measured by RT-qPCR. Enrichment of each RNA after capture with anti-SHARP antibody was calculated relative to the level of RNA captured with IgG control antibody. c, The enrichment of various lncRNAs after immunoprecipitation in pSM33 male cells—including Neat1, Malat1, Firre, and Tug1—are shown. d, The enrichment of various mRNA controls after immunoprecipitation in pSM33 male cells—including Oct4, Nanog, Stat3, and Suz12—are shown.