Single-Molecule Chromatin Dynamics Assay

Glass coverslips and microscopy slides containing drilled holes were cleaned by sonication in 10% alconox, acetone and ethanol, followed by treatment with a mixture of concentrated sulfuric acid to 30% hydrogen peroxide (3:1). The water-rinsed and dried coverslips and slides were then silanized using 2% (3-aminopropyl)triethoxysilane in acetone and assembled into flow cells, containing four channels each separated by double-sided adhesive tape. Pipette tips were inserted into the drilled holes and the channels were sealed with epoxy glue. A solution of 100 mg ml⁻¹ mPEG(5000)-succinimidyl carbonate containg 1% biotin-mPEG-succinimidyl carbonate was infused into the channels and reacted for 3 h, resulting in efficient passivation of all exposed surfaces in the channels. The channels were extensively washed using water and buffer T50 (10 mM Tris, 50 mM KCl) buffer before proceeding to experiments. For chromatin immobilization, 0.2 mg ml⁻¹ neutravidin solution was infused using a high-precision syringe pump and incubated for 5 min, followed by extensive washes with T50. Then, 500 pM chromatin arrays in T50 buffer were injected into the neutravidin treated flow chamber for 5 min, followed by a wash with T50 and imaging buffer (50 mM HEPES, 130 mM KCl, 10% glycerol, 2 mM trolox, 0.005% tween-20, 3.2% glucose, glucose oxidase/catalase enzymatic oxygen removal system). Chromatin coverage was observed in the TIRF microscope (Nikon Ti-E) by fluorescent emission in the far-red channel upon excitation by a 640-nm laser line. Dynamic experiments were initiated by infusion of 1 nM Atto532-labelled HP1α or 0.5 nM HP1α_cdm in imaging buffer. All proteins were freshly diluted from a 100 nM stock and immediately injected to avoid changes in concentration due to adsorption to tube walls. HP1α dynamics were observed in the yellow/orange channel using a 530 nm laser line for excitation at 20 W/cm² using an EMCCD camera (Andor iXon) at 20 frames/s for a 25 × 50 μm area at a resolution of 160 nm/pixels. Every 200 frames, an image of the chromatin positions in the far-red channel was recorded for drift correction.