Calibrated nuclear RNA sequencing (cnRNA-seq)

For cnRNA-seq, 1 × 10⁷ ESCs (untreated and OHT-treated) were mixed with 4 × 10⁶ Drosophila SG4 cells in 1× PBS. Nuclei were isolated in 1 mL HS lysis buffer (50 mM KCl, 10 mM MgSO₄.7H₂0, 5 mM HEPES, 0.05% NP40 [IGEPAL CA630], 1 mM PMSF, 3 mM DTT, 1× PIC [Roche]) for 1 min at room temperature, and then recovered by centrifugation at 1000g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris at pH 8, 3 mM MgCl₂). Nuclei integrity was assessed using 0.4% Trypan blue staining (Thermo Scientific). Pelleted nuclei were resuspended in 1 mL of TRIzol reagent (Thermo Scientific), and RNA was extracted according to the manufacturer's protocol, followed by treatment with the TURBO DNA-free kit (Thermo Scientific) to remove any contaminating DNA. Quality of RNA was assessed using the 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). RNA samples were depleted of rRNA with the NEBNext rRNA depletion kit (NEB) prior to preparing cnRNA-seq libraries using the NEBNext Ultra (for Bap1^fl/fl and PRC1^CPM ESCs) or Ultra II (for PRC1^CPM;Bap1^fl/fl ESCs) Directional RNA library preparation kit (NEB). To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was saved to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 15 min using the BioRuptor Pico (Diagenode), shearing genomic DNA to an average size of <1 kb. Libraries from sonicated genomic DNA were constructed as described above for cChIP-seq. Both cnRNA-seq and gDNA-seq libraries were sequenced as 80-bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicate.