To begin, add 500 microliters of protein-free image buffer to channel 3 of the flow cell. Mix two nanomolar of Saccharomyces cerevisiae NAP1 and two to five nanomolar of LD655-labeled H4 histone octamer with 500 microliters of 1X HR buffer. Add this mixture to channel 4 of the flow cell.
Flow all the channels at 1.0 bar for 30 seconds to flush them with samples. Set the flow to 0.2 bar, and move the optical traps to channel 1 to catch a suitable pair of streptavidin-coated beads. Next, move the optical traps to channel 3.
Turn off the flow, and conduct a forced calibration for the bead pair. Turn on the red confocal laser, and calibrate the imaging area. After setting the flow to 0.2 bar, move the optical traps to channel 2 to catch biotinylated DNA.
Then move the optical traps to channel 3, and stop the flow. Increase the distance between the optical traps to stretch the DNA, and monitor the associated forced distance curve to confirm the presence of a single DNA tether. Adjust the distance between the optical traps so that the DNA tension reads approximately one piconewton.
Turn on the line scanning to start collecting a kymograph with the red laser on. Then move the optical traps to channel 4 with the flow off to form nucleosomes along the DNA tether. For unwrapping nucleosomes, move the optical traps to channel 3.
Setting the force reading to 0 and speed to 0.1 micrometers per second, move the optical trap 1 away from the optical trap 2. Mix 10 nanomolar of Cy3-labeled linker histone H1.4 to 500 microliters of image buffer and add to channel 5. After forming a DNA tether containing nucleosomes, turn on the green laser, in addition to the red laser, and move optical traps to channel 5 for realtime imaging of H1 binding to chromatin.
Nucleosome formation along the DNA tether was visualized as stationary red fluorescent foci on a 2D scan and kymograph. Photobleaching analysis showed one-step or two-step photo bleaching events, indicating the presence of mononucleosomes. In the absence of NAP1, histone octamers bound DNA, but remained mostly unwrapped, indicated by the constant tension.
Using Cy3-labeled H2A, similar nucleosome assembly results were obtained, as with LD655-labeled H4.Nucleosome unwrapping increased tether distance over time, visible on the kymograph, and generated forced distance curves with characteristic rips that denote the unwrapping of individual nucleosomes. Linker histone H1 binding to chromatin was indicated by green fluorescent foci with dual color stationary trajectories representing the binding of H1 to nucleosomes, and green jagged trajectories representing diffusing H1 trajectories.
