Quantification of DSBs by immunofluorescence - Images & masks.

An atlas of endogenous DNA double-strand breaks

arising during human neural cell fate determination 

Abstract: Naturally occurring endogenous DNA double-strand breaks (DSBs) have been detected in neuronal cells and implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Here, we describe a genome-wide atlas of endogenous DSBs mapped by suspension Breaks Labeling In Situ and Sequencing (sBLISS) in proliferating neuroepithelial stem (NES) cells in culture, neural progenitor cells (NPC) after 6 days of NES differentiation towards neurons, and post-mitotic neurons (NEU) after 35 days of differentiation. 

This item contains imaging files from 53BP1 immunostaining performed on 10 fields of view image stacks per sample, each consisting of at least 48 focal planes spaced 0.2 µm apart, aiming at imaging at least 150 cells for three different stages of neuronal differentiation. 

DSB marker 53BP1 is reperesented by A488 fluorescence signal while the dapi channel is displaying HOECHST intensity. Image files follow naming structure Experiment_Sample_FoV as follows: 262_NES_001. 

Masks were generated as described on Github (https://github.com/elgw/sci_data_20220516).  The images were processed for quantification as described here in brief: Individual cells were segmented to generate 2D nuclei segmentation masks using the Otsu’s method to find a global threshold of the fluorescence intensity in the DNA channel, for each field of view. We then measured the fluorescence intensity in the channel corresponding to 53BP1 by integrating the images axially over the segmentation masks, using the mid 48 slices in each z-stack. When the images had more than 48 slices, we selected a range of z-values where the corresponding gradient magnitude was as high as possible (this corresponds to performing an axial centering around the middle section of the nuclei).