Imaging slides

Cells were plated on imaging slides (Ibidi), fixed with 4% paraformaldehyde, washed twice with PBS, and permeabilized with 70% ethanol overnight at 4 °C. After two washes with PBS and pre-hybridization solution (10% deionized formamide (Merck Millipore), 2x SSC), slides were incubated with 50 μl hybridization solution containing 2x SSC, 10% formamide, 50 μg competitor E. coli tRNA (Roche Diagnostics), 10% dextran sulfate (VWR), 2 mg/ml BSA (UltraPure; Life Technologies), 10 mM vanadyl-ribonucleoside complex (NEB) and 1 ng/μl smFISH probes for 6 h at 37 °C. Afterwards, slides were washed twice with pre-hybridization solution at 37 °C, then twice with PBS with subsequent mounting with ProLong® Gold Antifade Reagent with DAPI. Slides were imaged after 12 h when the mounting medium was fully cured on an Axio Observer.Z1 inverted epifluorescence microscope equipped with a × 63/1.4 Plan-APOCHROMAT objective (Zeiss).
Probe Designer software by Biosearch Technologies was used to design probes for hNEAT1 5′ segment and mNEAT1 middle segment, both conjugated to Quasar®670 fluorescent dye. Sequences are listed in Additional file 5: Table S4. Probes for hNEAT1 middle segment, mNEAT1 5′ segment, and MALAT1 (all conjugated to Quasar®570) were pre-designed by Biosearch Technologies.

Cells were grown on imaging slides (Ibidi), washed three times with PBS, and fixed with 4% paraformaldehyde (Sigma) in PBS for 10 min, followed by three washes using PBS. After permeabilization using 0.5% Triton-X-100 (Sigma) in PBS at 4 °C overnight and three washes with PBS, slides were blocked with 0.1% Triton-X-100 and 1% FBS in PBS for 1 h at room temperature. Incubation with primary antibodies was performed at 4 °C overnight. After three washes with PBS, slides were incubated with the species-corresponding secondary antibodies (Thermo Fisher Scientific) for 2 h at room temperature in the dark and washed three times with PBS afterwards. The samples were mounted with ProLong® Gold Antifade Reagent with DAPI (Thermo Fisher Scientific) on a coverslip and imaged with an Axio Observer.Z1 inverted epifluorescence microscope (Zeiss) equipped with a × 10/0.3 Plan-NEOFLUAR objective (Zeiss). Primary antibodies were diluted 1:100 unless stated otherwise and secondary antibodies 1:1000 in blocking buffer. Primary antibodies that were used in this study are listed in Additional file 5: Table S2.