MOESM4 of DNA replication dynamics of vole genome and its epigenetic regulation
2019-03-14T05:00:00Z (GMT) by
Additional file 5. Schematic rationale of single steps for mask generation used for quantification of nuclear PTM levels in untreated and treated/targeted cells. Confocal images were obtained using an UltraVIEW VoX spinning disk system (PerkinElmer, Massachusetts, USA) on a Nikon Ti microscope equipped with an oil immersion Plan-Apochromat x60/1.45 numeric aperture objective lens (pixel size in XY= 112 μm, Z-step 0.3 μm). For the calculation of mean DAPI and mean PTM intensities (H3K9ac, H4K8ac, H3K27me3, H3K9me3) in the whole nucleus, at the heterochromatic block at the X chromosomes or in the whole nucleus excluding the X, mid-nuclear sections of the DAPI and GFP channel were used to generate nuclear, X and exclusion masks, respectively. Images were processed using a median 3D filter and were threshold in four successive steps. For the generation of the binary masks, all pixels below the final threshold were set to 1, for both masks, respectively. Total PTM level values overlapping with the respective mask were calculated and divided by the total number of pixels corresponding to the area of measurement. To automate this analysis procedure, a routine was written in the programming language Python ( https://code.google.com/archive/p/priithon/ ). Mean values were measured and normalized to either untreated or untargeted samples.