Supplementary Figure 5: Centromeres are late replicating with CENP-A remaining tethered locally by continued binding to the CCAN complex.

(a) FACS analysis of DNA content showing the synchronization efficiency of CENP-A^TAP HeLa cell line across S phase. The experiment was repeated independently twice with similar results. (b) Genomic DNA of cells labelled for 1 hour with BrdU was sonicated prior to the BrdU immunoprecipitation and fragments of 200–800 bp were obtained. The experiment was repeated independently twice with similar results. Unprocessed images of DNA gels can be found in Supplementary Fig. 6. (c) Quantitative real-time PCR for MRGPRE and MMP15 genes, previously reported to replicate early (ref⁹ and ENCODE Repli-seq). (d) Quantitative real-time PCR for HBE1 and Sat2¹⁰ genes, previously reported to replicate late (ref¹¹ and ENCODE Repli-seq). (e) Quantitative real-time PCR for α-satellite DNA. Data shown in c-e are from two biologically independent experiments. Source data for c-e can be found in Supplementary Table 4. (f) MNase digestion profile showing the nucleosomal DNA length distributions of bulk input mononucleosomes (upper panel) and purified CENP-A^TAP following native ChIP at early S and mid-S phase. The experiment was repeated twice independently with similar results. (g) CENP-A ChIP-seq raw mapping data spanning the whole of cen18 at G1, mid-S phase and G2, and BrdU repli-seq at early S (S1), mid-S (S4) and late S/G2 (S7). SICER peaks are denoted as black lines underneath the raw mapping data. The experiment was repeated twice independently with similar results. Centromere reference location, red. CENP-B boxes, orange. Scale bar, 2Mb. (h) Overlap degree between CENP-A G1 and mid-S at α-satellite HORs single copy variants. (i) Ethidium Bromide stained DNA agarose gel showing MNase digestion profile of bulk chromatin used for mass spectrometry identification of proteins associating with CENP-A^TAP chromatin (left panel) and for CENP-A^TAP co-immunoprecipitation experiment (right panel). Mass spectrometry was performed once and co-IP was performed twice with similar results. Unprocessed images of DNA gels can be found in Supplementary Fig. 6. (j-n) CENP-A^TAP immunopurification followed by mass spectrometry identifies association with CENP-A chromatin of DNA replication related proteins (j,k), chromatin remodelling factors and nuclear chaperones (l), histones (m) and centromere and kinetochore proteins (n).