DT40 Cell cultures synchronously entering mitosis were analyzed by Hi-C, imaging and proteomics to determine the structure of chromosomes. Hi-C data were used to quantify chromosome compartmentalization and to derive relationships between contact frequency P and genomic distance s. Coarse grained models and equilibrium polymer simulations were performed to test models of prophase and prometaphase chromosome organization against Hi-C data, and to identify best fitting parameters for size of loops, helical turn and pitch, linear density (Mb/micron chromosome length). Imaging of chromosome dimensions and condensin localization were performed to validate model predictions. Cell lines expressing condensin subunits fused to auxin-inducible degron domains were used to efficiently deplete these subunits prior to cells entering mitosis. Hi-C and imaging analysis were then performed to assess the effects of depletion of condensins on mitotic chromosome formation. Detailed procedures for all methods are described in the Supplementary Materials.