Matlab and Python scripts to analyze phosphorylation and morphology of RNA Pol II clusters

MatLab and python scripts for fluctuations of phosphorylation and morphology of RNA polymerase II clusters. <strong>Requirements</strong> N2v package should be installed. The installation guide can be found in https://github.com/juglab/n2v <strong>Analysis Procedure</strong> Download the required data in Nikon .nd2 format. You need all data from recruited RNA polymerase II Serine 5 (Pol II Ser5P ) and RNA polymerase II Serine 2 (Pol II Ser2P ) in live sphere stage zebrafish embryos, visualized with antibody fragments (Fab). Images should be placed in a folder named “image”. Train the n2v network on a single image which is recorded with the same configuration as the live images. You can run the python script on Google Colab to save time and process resources. The script can be found at https://colab.research.google.com/drive/1KLJcvng36JiPlGH2GWaO2VxR2bppJ0xc?usp=sharing. The patches which are given to the network are of size (16, 64, 64), while 4/5 of the patches were used to train the network and 1/5 of all patches were used for the validation. You would better leave the network to be trained on each image at least for 70 epochs with the neighbourhood radius = 10. ... mehrYou can also use the original script provided at https://github.com/juglab/n2v/blob/master/examples/3D/01_training.ipynb with this configuration. Python script <strong>TimeLapsReconstruction.py </strong>reads a single .nd2 image and reconstruct the ser5P images by n2v network. An already-trained network configuration is in <strong>"model.zip"</strong>. You can easily replace it with your own n2v model. <strong>TimeLapsReconstruction.py </strong>put all the reconstructed ser5P images and the original ser2P images together and generate a single TIF file. If you would like to skip the previous steps and directly go to step 3 you can download the <strong>EmbryoC_50ms_042_reconstructed.tif</strong> and place it in the folder named "image". Matlab script <strong>morphology_fluctuation_analysis.m </strong>reads the TIF file generated in the previous step and segments Pol II clusters and tracks clusters over time. For each focus mean intensity of Ser5P, the mean intensity of Ser2P, solidity and elongation is calculated and tracked over time. It shows how these quantifications change over time. The script later calculates the cross-correlation analysis of the temporal coordination of Pol II Ser5P intensity, Pol II Ser2P intensity, and solidity with elongation. Comments are provided n the script and the code is easily understandable. There are some functions that are called through <strong>morphology_fluctuation_analysis.m </strong>script. The required functions are in a folder named “required function”. You should download and unzip the folder and add it to your Matlab.