Supplementary Figure 4: Reproducibility of the HRF quantification results from independently repeated experiments.

Left: PAGE gel of HRF experiments for G. gallus nucleosome reconstituted on a 601 sequence. Experimental details can be found in ref. ³⁶. Briefly, histones were extracted from G. gallus erythrocytes and nucleosomes were reconstitutes on a 601 Widom positioning sequence with a radioactive label on the 5ʹ end. After hydroxyl-radical footprinting of nucleosomes, DNA was extracted and subjected to denaturing PAGE followed by radioactive signal acquisition using phosphorimaging. The lanes on the gel correspond to the following reaction products: TS - experiments for the top DNA strand, BS – experiments for the bottom DNA strand. N represents HRF reaction products for the nucleosomal DNA, and A, G and T are the sequencing lanes and mark the products of primer extension using Taq DNA polymerase and terminating dideoxynucleotide triphosphates (ddATP (A), ddTTP (T), and ddGTP (G), respectively). Experiment A and Experiment B labels mark the lanes resulting from two independent HRF experiments performed on the same sample of nucleosomes. The blue lines indicate the area of the gel used to extract gel lane profiles. Right: Comparison of profiles extracted by HYDROIDexp from independently repeated HRF experiments. The y-axis represents the normalized values of DNA cleavage frequency. When plotting two profiles on one plot the “every” method from HYDORID package was used, which simply normalizes every profile to its maximum value. Zero on the x-axis marks the nucleotide positioned on the central dyad symmetry axis of nucleosome.