Image-Based Quantitative Single-Cell Method Suggests Increase of Global Chromatin Accessibility in Tumor Compared to Non-tumor Cell Lines
Description
In this study we tested the hypothesis that staining of fixed cells with several DNA ligands may report chromatin accessibility in each nucleus. These are tables generated by Gen5 automated imaging software using Cytation 5 Imager. Each table contains raw data used to generate plots for each figure in the manuscript with the same name as a dataset.
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For all imaging experiments, cells were plated into 96 well black plates with clear bottoms (Greiner Bio-One, Monroe, NC, cat # 655090), 5000 cells per well. For flow cytometry cells were plated in 6 well plate at 100,000 cells per well. Next day cells were treated with CBL0137 for 10-60 min or epigenetic drugs for 24 hours. After treatment medium with drugs was removed and cells were fixed with 4% paraformaldehyde (PFA) in PBS with 0.1% Triton X100 or 100% ice-cold methanol for 10 min at room temperature. For flow cytometry cells were first trypsinized, resuspended in medium with 5% FBS, washed from medium with PBS and fixed in 4% PFA. Then fixatives were removed, and cells were stained with DNA ligands with or without RNAse A (100 g/ml). Unless otherwise stated, the following concentrations of DNA ligands were used Propidium Iodide (PI) – 1 microg/ml, DAPI and Hoechst 33342 – micromole, SYBR Green – 50 micromole (1:100 dilution of solution provided by vendor). Plate was left in staining solution overnight at room temperature to allow RNA digestion. Plate imaging was done using Cytation 5 automated imager (Biotek/ Agilent Technologies, Santa Clara, CA). We used a 4X and 10X objectives, 4 images or 9 images per well of 96-well plate, four replicate wells per condition. Data was obtained with 4X objective. Cytation 5 built-in Gen5 software (Agilent) creates a montage of images taken from the same well. We used ‘autofocus’ mode, which ensures that each image is in focus independently of potential irregularities in plastic thickness. Data collection was done from ~300-5000 objects per well. Object masking was done to include only nuclei. For the same DNA stain, we set the same exposition time and object masking parameters for the whole experiment. If experiment consisted of several plates, each one of them included positive/negative controls for comparison. This serves as intrinsic normalization for the data collection. The data we collected and included into the manuscript included (i) ‘size’ -the longest linear dimension of an object/nucleus (in m), (ii) ‘area’ – the square of an object/nucleus (in m2). These parameters are specific to the camera (CMOS), and the objectives used, and are gained from an image as the number of pixels per object (or per the longest dimension of an object for ‘size’) multiplied by the square (or by length for ‘size’) of each pixel. In our case, for images taken with 4x objective the pixel length was 1.28 m and the area - 1.64825 m2. (iii) ‘Mean’ - is the intensity of fluorescence of one pixel of an object, averaged for all object pixels. (iv) ‘Integral’ - is the sum of the fluorescent intensities of all pixels from an object.