Stellaris RNA FISH probes (Biosearch Technologies, CA, USA) were used, with 48 × 20 mer fluorophore conjugated oligos tiling the length of the target transcript. GAPDH and NEAT1 probe sets were supplied as predesigned controls conjugated to Quasar 570 fluorophores. Staining was carried out as described in the Stellaris protocol for adherent mammalian cells, but adapted for cells grown on coverslips using 15% rather than 10% formamide for hybridization, which was found to reduce background signal. GAPDH and NEAT1 probe sets were used at 50 nM. IPSC-derived matured neurons were imaged using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany) with manufacturer provided ZEN imaging software. Presented images are maximum intensity projections of 20 Z-stack slices taken with a standard 1 AU slice depth and 0.5 AU intervals, capturing the entire volume of the cell. Other than linear contrast/brightness adjustments the images are unmodified. Unstained cells used as a negative control showed no fluorescence signal using identical imaging settings.
