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1 DOI: 1.1/ncb2918 Supplementary Figure 1 (a) Biotin-dUTP labelling does not affect S phase progression. Cells synchronized in mid-s phase were labelled with biotindutp 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 gh2ax 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 2 minutes and fixed directly (nascent) or left for 2 hours 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 biotindutp intensity per cell (right) after 5, 1, 2 and 4 minutes of labelling. Horizontal lines represent the median, ****p <.1, *p =.18, n.s. p =.2918 (unpaired t test, 18 < n). Statistics source data is available in Supplementary Table 4. (e) Western blot analysis of NCC pull-downs after 5 and 1 minutes pulse-labelling with biotin-dutp. Labelling times of >5 minutes 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. 1

2 a LIGHT biotin-dutp Lys Arg Release 3 hrs from thymidine block 2 min HEAVY biotin-dutp Lys8 Arg1 Release 3 hrs from thymidine block 2 min Chase 2 hrs Nascent chromatin Mature chromatin Mix heavy and light in 1:1 ratio Mass spectrometry analysis by Orbitab and quantitation by Maxquant b c Heavy - Light biotin-dutp positive cells (%) Heavy Light Cell counts (% of Max) G1 G2 Time of biotin-dutp labelling G1 G2 Time of harvesting d e log 2 protein ratios (N/M) 8 Replicate A or C Replicate B Count 2, 1,5 1, 5 Fold Difference Histogram Rep A vs B: all proteins Rep A vs B: replication factors Rep C vs B: all proteins Rep C vs B: replication factors Median fold difference between replicates log 2 protein ratios Supplementary Figure 2 (a) Experimental design for NCC-SILAC. Two independent cultures grown in heavy and light amino acids were released into S phase from a single thymidine block. Cells were labelled with biotindutp for 2 minutes 3 hours after release into S phase. Light cultures were left to progress in S phase for 2 hours after labelling, while heavy cultures were harvested immediately. Synchronization and release of heavy cultures were shifted 2 hours with respect to light cultures, such that they could be cross-linked and processed in parallel for NCC. After pull-down, the heavy and light samples were washed stringently and mixed prior to SDS-PAGE and mass spectrometry. (b) Biotin-dUTP incorporation scored by immunofluorescence verified equal labelling efficiency in heavy and light cultures. Approximately 25 cells were counted. (c) Cell cycle profile of heavy and light cultures verified matching synchronization at the time of the labelling (left) and that light cultures had progressed to late S phase at the time of harvest (right). (d) Scatter-plot comparing log 2 SILAC ratios between replicates and (e) histogram of median of pairwise log 2 fold difference of SILAC ratios between three replicates, illustrating reproducibility of the NCC- SILAC technology. 2

3 Supplementary Figure 3 (a) Nascent chromatin enrichment of factors proposed to deal with DNA-RNA duplexes and protein degradation at the fork 2, 5. (b) Nascent chromatin enrichment of origin recognition and licensing factors. (c) Coverage of the PCNA interactome by NCC-SILAC. Bars illustrate factors identified in NCC relative to those reported in the literature, numbers are indicated in brackets. Functional groups of PCNA interactors are according to Moldovan et al., 27. For details see Supplementary Table 2. (d) Analysis of factors enriched on nascent chromatin by western blot. For comparison the log 2 nascent chromatin enrichment is indicated. (e) Nascent chromatin enrichment of lysine and arginine methyltransferases (violet) and demethylases (grey). (f) Nascent chromatin enrichment of RNA polymerases. Only unique subunits are shown. All Nascent chromatin enrichments are presented as in Figure 1h. 3

4 Supplementary Figure 4 Box plot of nascent chromatin enrichment of the individual classes defined in Figure 5a. 4

5 a c Distribution (%) Nuclear Cytoplasmic both FAM8A ATAD2B d b Colocalization with PCNA (Pearson coefficient) FAM8A ATAD2B Distribution (%) Nuclear Cytoplasmic both No signal PTCD3 RPL29 KIAA391 MPST Colocalization with PCNA (Pearson coefficient) No signal PTCD3 No signal RPL29 KIAA391 MPST Supplementary Figure 5 Analysis of GFP- or FLAG-tagged proteins in U-2-OS cells stably expressing RFP-PCNA. Localization pattern (a, c) and co-localization with PCNA measured by Pearson coefficient (b, d) were scored after pre-extraction. The Pearson coefficient is calculated for individual nuclei and shown in a box plot (n 9). Horizontal line represents the median. 5

6 a b c FAM8A PIP Box APIM FLAG DAPI WT APIM 2 μm PCNA patterns (%) No PCNA Early S PIPmt Mid S H2B Late S d e f EdU MCM2 GFP PCNA EdU GFP PCNA-RFP DAPI WT PIPmt H2B PIPmt WT Early S H2B G1 Mid-late S G2 2 μm 2 μm 2 μm g U-2-OS sicontrol si-1 si-2 1x 1x 1x * Ponceau S h EdU intensity in PCNA + cells (a.u.) **** **** sicontrol si-1 si-2 i PCNA intensity in EdU + cells (a.u.) 4 2 **** **** sicontrol si-1 si-2 Supplementary Figure (a) Conservation of the PIP box and FAM8 APIM in mammals 57. (b) Localization of FLAG-tagged FAM8A wild type (WT) and APIM mutant (APIMmt). (c) PCNA patterns in cells transfected with WT or PIPmt. H2B was used as a control. The mean and of three independent experiments is shown with error bars representing s.d., 8 < n < 5. (d) Representative EdU and MCM2 patterns used to analyse cell cycle distribution in Figure 7d. The EdU pattern distinguishes S phase stage (early, mid or late), while the MCM2 pattern distinguishes EdU negative cells in G1 and G2. (e) Cells expressing the PIPmt show a low level of EdU incorporation and strongly reduced loading of PCNA on chromatin. Note that the GFP- PIPmt does not localize to EdU foci, in contrast to the wild type protein. Transfected cells were labelled for one hour with EdU and co-stained with PCNA. Representative images are shown. (f) RFP-PCNA levels in a stable cell line transfected with WT and PIPmt. Representative images are shown with arrowheads marking transfected cells. (g-i) depletion in U-2-OS cells transfected with two independent sirnas for 48 hours. Western blot (g), 2x indicates the double amount of sample loaded in 1x. (*) unspecific band. Quantification of EdU incorporation (h) and chromatin-bound PCNA (i). Dot plot of PCNA intensities in EdU positive cells are shown. Similar results were obtained in total cells population. One representative experiment is shown. Horizontal lines represents the median, ****p <,1 (unpaired t test, < n). Similar results were obtained using a SMART pool (Dharmacon) directed against (data not shown). Statistics source data are available in Supplementary Table 4.

7 Alabert et al. Supplementary Figure 7 Histone H4 H4K12ac PCNA Figure 1f Histone H3 Histone H2B Figure 4a H4K5ac H3K9me1 H3k27me3 H3k9me3 H3K27me1 Histone H4 H3K9me3 H4K5ac H4K5ac Figure 4b H4K5ac H3K27me3 β-actin 49 Histone H4 Histone H3 Supplementary Figure 7 Uncropped Western blots. 7

8 Figure f 2 49 Figure g Histone H3 PCNA 35 S Figure g 35 S Coomassie Figure 7g 98 2 Ponceau Supplementary Figure 7 continued Uncropped Western blots. 8

9 Histone H3 H4K5ac PCNA Ponceau Supplementary Figure 1e Supplementary Figure g Histone H4 HP1γ DNMT1 CTF18 CAF1 p PCNA Supplementary Figure 3d Supplementary Figure 7 continued Uncropped Western blots. 9

10 Supplementary Table Legends Supplementary Table 1. List of the 3995 factors quantified by NCC-SILAC. Column S indicates the replicate experiment(s) (A, B and/or C) in which a protein was quantified. Supplementary Table 2. List of known PCNA interacting factors and their NCC-SILAC enrichment. Categories are adapted from 23. Supplementary Table 3. List of chromatin proteins enriched in nascent chromatin (see Fig. 5d). Supplementary Table 4. Statistics source data. 1