Wide field deltavision elite microscope

Z stacks with 200–250 nm z-step capturing the entire cell volume were acquired with a GE wide-field DeltaVision Elite microscope with an Olympus UPlanSApo 100×/1.40-numerical aperture oil objective lens and a PCO Edge sCMOS camera using appropriate filters. The built-in DeltaVision SoftWoRx Imaging software was used to deconvolve the three-dimensional stacks. Maximum intensity projections were generated in Fiji and subjected for quantification using Fiji. To outline cell borders, we used manual segmentation. For an accurate manual cell segmentation, the saturation of the FISH channel was increased to visualize cytoplasm contours. Overlapping cells were not quantified. The brightness and contrast of each channel was adjusted. Overlapping exon/intron spots were considered as intron-retained transcripts, while exon only transcripts as spliced transcripts. Each imaging experiment was performed at least two times quantifying at least 50 cells across independently acquired datasets. For ActD treatment and mitosis, less cells/mitosis were quantified per treatment, as indicated in the figure legend. Analysis of z-stacked was additionally performed in 3D in Imaris to confirm that nuclear intron-retained transcripts were within the nucleus. γH2A.X foci were quantified with ImageJ plugin Spot Counter v.0.14 maintaining the same spot size between conditions.

We acquired z-stacks (200 nm z-step) capturing the entire cell volume for single-molecule RNA FISH, single-molecule RNA FISH/CMXR staining, 3xFLAG tag immunofluorescence/CMXR staining, and/or Tom20 immunofluorescence with a GE wide-field DeltaVision Elite microscope with an Olympus UPlanSApo 100x/1.40-NA Oil Objective lens and a PCO Edge sCMOS camera using corresponding filters. 3D stacks were deconvolved using the built-in DeltaVision SoftWoRx Imaging software. Maximum intensity projections of each image were subjected for quantification using Fiji.