Supplementary Figure 1

(a) Biotin-dUTP labelling does not affect S phase progression. Cells synchronized in mid-S phase were labelled with biotin-dUTP during a 5 min hypotonic shift (red) or incubated in PBS (blue), and analysed by FACS at the indicated times. (b) Biotin-dUTP labelling does not trigger DNA damage as measured by γ H2AX staining. Cells were treated with hydroxyurea (HU, 3 mM) for one hour as a positive control. (c) Co-localization between PCNA and biotin-dUTP is lost during chromatin maturation. U-2-OS cells stably expressing PCNA-RFP were pulse labelled with biotin-dUTP for 20 min and fixed directly (nascent) or left for 2 h before fixation (mature). Cells not treated with biotin-dUTP are shown as a negative control. (d, e) NCC with shorter biotin-dUTP labelling times. (d) The percentage of biotin-dUTP positive cells (left) and the biotin-dUTP intensity per cell (right) after 5, 10, 20 and 40 min of labelling. Horizontal lines represent the median, ****p < 0.0001, p=0.0168, n.s. p= 0.2918 (unpaired t test, 108 < n). Statistics source data is available in Supplementary Table 4. (e) Western blot analysis of NCC pull-downs after 5 and 10 min pulse-labelling with biotin-dUTP. Labelling times of > 5 min are preferable for efficient incorporation of biotin-dUTP. The amount of starting material should always be adjusted according to labelling time in order to achieve sufficient material for a comprehensive proteomic analysis.