Image data description Color channels in the image data: First channel: DNA (Hoechst 33342) Second channel: Pol II Ser2P (Elongating RNA polymerase II, indirect immunofluorescence, STAR RED) Third channel: Pol II Ser5P (Recruited RNA polymerase II, indirect immunofluorescence, Alexa 594) Sample description Praimary cell cultures were prepared from pluripotent zebrafish embryos were collected at the sphere stage of development, treated with chemical inhibitors of transcription (Control, 500 mM Flavopiridol, 500 mM Triptolide, all for 30 min) and fixed overnight (2% formaldehyde in culutre media, 30 min, room temperature; followed by 8% formaldehyde for 15 min at room temperature). RNA polymerase in the recruited state (Pol II Ser5P) and the elongating state (Pol II Ser2P) were labeled by indirect immunofluorescence (permeabilization 0.5% Triton X-100 in PBS 15 min room temperature, 30 min blocking 4% BSA in PBST, rat IgG anti-Pol II Ser5P & rabbit IgG anti-Pol II Ser2P in 4% BSA in PBST overnight 4°C, anti-rat Alexa 594 & anti-rabbit STAR RED in 4% BSA in PBST overnight 4°C). Mounted in VectaShield H-1000 with 2 µM Hoechst 33342 added for fluorescent DNA labeling. Scan of lab book page is included in the repository. The data set contains images obtained from two samples for each condition. Imaging Microscopy images acquired using VisiTech iSIM with dual camera setup. Objective Nikon CFI SR HP Apo TIRF 100XAC Oil, color channels acquired in a sequence to reduce overlap (DNA + Ser2P acquired simultaneously on two camerase, Ser5P acquired after on a single camera), z-stack settings optimized to ensure reliable xyz alignment of channels. Imags were cropped to the region with best signal and resolution in the DNA channel, same region used throughout the entire dataset. all imaging settings were kept unchanged over the course of acquisition, all images acquired in a single session of 4 hours. Advice for image processing Images can be loaded for processing with the OME bioformats importer. An import script for MatLab is available through the Hilbert lab: https://github.com/lhilbert/NuclearObjects_ImageAnalysis Author contributions AN & MS provided embryos, carried out cell culture, and prepared samples, LH performed microscopy