Preparation of Nitrocellulose-Coated Microfluidic Chambers

Sample chambers were prepared as previously described^{27 (link)} with the listed modifications. In brief, microscope coverslips were coated with a thin layer of nitrocellulose by spin coating. Coated coverslips were rinsed and stored in a clean plastic container for 48 h or more before being assembled into a microfluidic sample chamber using inert silicone high vacuum grease (Dow Corning 1597418). In contrast to previous work, nitrocellulose-coated coverslips were used for both the top and bottom surfaces of the sample chamber instead of only the bottom surface.
Tethers for MT experiments were formed as previously described^{27 (link)} with some modifications. First, a nitrocellulose coated sample chamber was incubated with 10 ng/µL anti-digoxygenin (Vector Labs MB-7000) in phosphate buffered saline (PBS) for 30 min. Next, 1 µm magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher 65601) coated with biotin and digoxigenin labeled DNA were bound to the surface to serve as fiducial markers. Then, 5 mg/ml β-casein (MilliporeSigma C6905) was incubated in the chamber for at least 1 h to passivate the surfaces. The DNA substrate of interest was then incubated at concentrations ranging from 1.5 pM to 10 pM for 15 min, followed by magnetic beads at a concentration of 20 µg/ml for 15 min. The buffer in the sample chamber was then exchanged for the topo reaction buffer.