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 biotin-dUTP for 20 min 3 h after release into S phase. Light cultures were left to progress in S phase for 2 h after labelling, while heavy cultures were harvested immediately. Synchronization and release of heavy cultures were shifted 2 h 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 250 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 log₂ SILAC ratios between replicates and (e) histogram of median of pairwise log₂ fold difference of SILAC ratios between three replicates, illustrating reproducibility of the NCC-SILAC technology.