Supplemental Material for Toraason et al., 2021

File S1 contains a step-by-step protocol for the Gonad Analysis Pipeline at the time of this manuscript’s publication. Figure S1 displays bar plots representing the proportion of nuclei identified from each region of the germline by the Whole Gonad Pipeline. Figure S2A displays plots of the simulated ‘germlines’ and ‘nuclei’ utilized to validate the Gonad Linearization algorithm. Figure S2B displays a density plot of the proportion of ‘nuclei’ in simulated ‘germlines’ which were accurately assigned to central line segments. Figure S2C displays a plot comparing the rank order of simulated ‘nuclei’ correctly assigned to central line segments within simulated ‘germlines’ to their known simulated rank order. Figure S2D displays density plots showing the relative deviation of simulated ‘nuclei’ from their known simulated positions relative to the alignment performed by the Gonad Linearization algorithm. Figure S3D displays dot plots assessing the association of DSB-2 staining intensity and RAD-51 focus counts in germlines analyzed by the Gonad Analysis Pipeline. Figure S4AB displays immunofluorescence images on whole gonads of spo-11(me44) and rad-54(ok615) whole germlines. Figure S4C displays dot plots assessing RAD-51 focus counts per nucleus in spo-11(me44) and rad-54(ok615) whole germlines analyzed by the Gonad Analysis Pipeline. Figure S5 displays dot plots assessing “bright” MSH-5 foci counts per nucleus in germline analyzed by the Gonad Analysis Pipeline. Figure S6A displays immunofluorescence image of MSH-5 and RAD-51 staining in germline. Figure S6B displays a bar graph of the frequency of colocalization between MSH-5 and RAD-51 foci. Figure S6C displays a density plot of the distance between a MSH-5 focus and the nearest RAD-51 focus considered colocalized versus not colocalized.