Figure 6.

Solid-state nanopore analysis of CENP-A chromatin reveals it to have the physical characteristics of nucleosomes. (A) Experimental design of chromatin translocation through a solid-state 20-nm-wide nanopore. (B) Conductance drop (ΔG) measurements during the translocation of (top, red) in vivo affinity-purified CENP-A^TAP–containing chromatin particles (total events = 3,096), in vivo affinity-purified H3.1^TAP-containing chromatin particles (middle, green; total events = 4,141), and (bottom, purple) in vitro–reconstituted (CENP-A/H4)₂ tetrasomes (total events = 383). n = 2 from two independent datasets. Error bars represent SD. A fitted Gaussian distribution is overlaid in black. Fits were performed by the Levenberg–Marquardt algorithm to reduce the χ² value. The reduced R² values for the three fits were 0.94, 0.954, and 0.961. ΔG values represent the mean of the fitted Gaussian distribution, and the error bars are the width of the Gaussian fits. Vertical dashed line is the peak ΔG value for the tetrasome, as measured by the Gaussian fits. (C) Summary of the chromatin types measured in this study and previously (Soni and Dekker, 2012) using solid-state nanopore, along with their predicted molecular masses (with 4.5 kD added to each tagged histone to account for the S-peptide tag remaining after TEV cleavage). In vitro H3.1 nucleosomes were reconstituted on a 344-bp 601 nucleosome positioning sequence.