Microscopy-based assessment of RNA polymerase II clusters during differentiation of cultured mouse embryonic stem cells

This repository contains the data and MatLab analysis scripts of the analysis of RNA polymerase II clusters over the course of differentiation of cultured mouse embryonic stem cells (mESCs). The cells were induced towards differentiation by either withdrawal of lineage-inhibiting factors (-LIF) or by RHB treatment. Both treatments were carried out in duplicate in independent experiments. Time course data were obtained by collection of cells at 3, 6, 12, 24, and 48 hours following the beginning of induction. Control data from unindexed cells were obtained at 24 and 48 hours. Cells were cultured, treated, collected, and fixed at University Medical Center Göttingen by Yomna Gohar, Priya Kumar, and Carmelo Ferrai. Fixed cells were fluorescently labeled and microscopy images were recorded and analyzed at Karlsruhe Institute of Technology by Lennart Hilbert. To analyse date data, the raw image data are first extracted into MatLab-native files using the MultiPosition_extraction_nd2.m script. The actual analysis is then carried out using the ClusterAnalysis.m script. Example microscopy images were produced using the ExampleImages.m script. The extracted data can be reviewed using the ReviewExtractedStacks.m script. This is the main repository, containing the image analysis scripts alongside the links to the raw image data used in the analysis. The raw data are too large to be stored within this single repository, and therefore can be found at the following additional addresses. Differentiation by withdrawal of lineage-inhibiting factors (-LIF): 10.5281/zenodo.8013281 Differentiation with addition of RHB for neuronal fate induction (RHB): 10.5281/zenodo.8013307