The 193 bp 601 DNA fragment was amplified by a PCR reaction (62 (link), 63 (link)). The nucleosomes were assembled with the salt dialysis method described above. The reconstituted nucleosome was dialyzed into buffer XL (80 mM PIPES-KOH [pH 6.8], 15 mM NaCl, 60 mM KCl, 30 % glycerol, 1 mM EGTA, 1 mM MgCl2, 10 mM β-glycerophosphate, 10 mM sodium butyrate). H1.8-GFP was mixed with nucleosome with a 1.25 molar ratio in the presence of 0.001 % poly L-glutamic acid (wt 3,000–15,000) (Sigma-Aldrich) and incubated at 37 °C for 30 min. As a control nucleosome sample without H1.8-GFP, the sample without H1.8-GFP was also prepared. The samples were then crosslinked adding a 0.5-time volume of buffer XL containing 3 % formaldehyde and incubating for 90 min on ice. The crosslink reaction was quenched by adding 1.7 volume of quench buffer (30 mM HEPES-KOH (pH 7.4), 150 mM KCl, 1 mM EGTA, 10 ng/μL leupeptin, 10 ng/μL pepstatin, 10 ng/μL chymostatin, 10 mM sodium butyrate, 10 mM β-glycerophosphate, 400 mM glycine, 1 mM MgCl2, 5 mM DTT). The quenched sample was layered onto the 10–25 % linear sucrose gradient solution with buffer SG (15 mM HEPES-KOH [pH 7.4], 50 mM KCl, 10–22 % sucrose, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml chymostatin, 10 mM sodium butyrate, 10 mM β-glycerophosphate, 1 mM EGTA, 20 mM glycine) and spun at 32,000 rpm (max 124,436 rcf) and 4 °C for 13 h using SW55Ti rotor in Optima L80 (Beckman Coulter). The centrifuged samples were fractionated from the top of the sucrose gradient. The concertation of H1.8-GFP bound nucleosome in each fraction is calculated based on the 260 nm light absorbance detected by Nanodrop 2000 (Thermo Scientific).