For validation of the correction method (Figs. 1 and 2), we designed a ~1-kbp construct in which two 522-bp dsDNA tethers were bridged by a hairpin structure (fig. S1A). The tethers were prepared by PCR using biotin- and digoxigenin-modified primers for surface and bead attachment, respectively. The hairpin consisting of a 30-bp stem and a 4-nt loop (24) was separately synthesized by annealing and ligation of oligos. The hairpin strand and tethers were assembled by enzymatic digestion and ligation through their cohesive ends and verified in agarose gels. For the constructs used in the measurements of persistence length (Fig. 4), dsDNA fragments of varying length (198 bp to 4 kbp) and GC content (30 to 64%) were prepared by PCR and ligated with oligos that collectively formed a hairpin structure at one end of the dsDNA backbone (fig. S1B). Further details are in Supplementary Materials and Methods. For CpG methylation, 510-bp constructs with a hairpin at the biotin side were methylated at their CpG sites via CpG methyltransferase M.SssI (New England BioLabs). For each construct, ~800 ng of DNA was mixed with 240 μM S-adenosyl methionine and 8 U of M.SssI in the standard reaction buffer (NEBuffer 2) and incubated for 10 hours at 37°C. For the verification of methylation, the products were digested with methylation-sensitive restriction enzyme Bst UI (New England BioLabs), and the cleaved segments, if any, were visualized on a 2% agarose gel. The degree of protection by methylation was measured to be >90% in all cases.

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