Microscopy-based analysis of the DNA and RNA polymerase II distribution inside nuclei of pluripotent zebrafish embryos

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

Pluripotent zebrafish embryos were collected at the sphere stage of development and fixed overnight (0.3X Danieu's media with 2% formaldehyde, 0.2% Tween-20, 4°C). RNA polymerase in the recruited form (Pol II Ser5P) and the elongating form (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:

One sample (8 embryos in the sample) with all three colors labeled (Main Sample)

One sample (3 embryos) in which the primary antibodies were omitted to allow assessment of cross-talk to the DNA channel (No Primary Antibodies)

One sample (1 embryo) in which no Hoechst was added to the mounting media to allows assessment of cross-talk to the immunofluorescence channels (No Hoechst)

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

Image analysis scripts

Scripts for the image analysis of the shapes of Pol II clusters in relation to transcription levels are provided as MatLab files. The MatLab script MultiPosition_extraction.m should be run as the first script, and requires that the bfmatlab toolbox from the Open Microscopy Environment is installed and added to the path permanently. Then, the script ReviewExtractedStacks.m was used to sort out images from the image folder that are clearly dominated by prominent miR-430 foci. Following this, the script AmphiphileExampleImages.m can be used to produce example images, and the script ClusterAnalysis_Amphiphile.m to carry out the actual image analyses. A figure prepared from the final image analysis is also provided for guidance.

Author contributions

AN & MS provided embryos and prepared samples, LH performed microscopy