RNA ISH was performed on tissue microarrays using an RNAscope 2.5 HD Detection Kit (RED, Advanced Cell Diagnostics) or an RNAscope Multiplex Fluorescent Reagent Kit according to the manufacturer’s instructions. For ISH, tissue sections were deparaffinized with xylene and 100% ethanol, incubated in a H2O2 solution for 10 min, heated in target retrieval buffer at 95° to 99°C for 20 min, and digested in protease solution for 30 min. The slides were then hybridized with a custom probe, Hs-LOC105372310-O2, in a HybEZ oven (Advanced Cell Diagnostics) at 40°C for 2 hours. After signal amplification and detection, the slides were dried in a 60°C dry oven for 15 min and mounted with EcoMount. Positive staining density was measured using a computerized imaging system composed of a Leica CCD DFC420 camera connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd.). The H-score scoring system was used, which evaluates staining intensity (0 to 3) and the percentage of positively stained cells (0 to 1), with a final score ranging from 0 to 3. For fluorescent ISH, cells were rinsed in PBS twice, fixed in 4% formaldehyde in PBS (pH 7.4) for 30 min at room temperature, incubated in a H2O2 solution for 10 min, and digested in protease solution for 10 min. The slides were then hybridized with a custom probe, Hs-LOC105372310-O2, in a HybEZ oven (Advanced Cell Diagnostics) at 40°C for 2 hours. After signal amplification, the slides were conjugated with TSA Plus Cy3 (PerkinElmer). The slides were then incubated with anti-ZMYND8 antibody overnight at 4°C. After incubation with the fluorescein-conjugated secondary antibody, the slides were mounted with ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole.

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