Bead-Loaded Fluorescent Fab Delivery

Cells were cultured in glass-bottom dishes (35 mm, 14 mm glass, Mat-Tek). The next day dye-conjugated Fabs were loaded into the cells through bead-loading^{22 (link),27 (link),29 (link),66 (link),67 (link)}, as follows: First, the fluorescent Fabs (CTD-RNAP2-CF640 and Ser5ph-RNAP2-Cy3, ~1 mg/mL, each) were mixed with PBS up to 4 μL in the cell culture hood. Second, the medium was removed completely from the dish and stored, and the Fab mixture was added to the center of the dish. Third, glass beads (106 μm, Sigma-Aldrich, G-4649) were immediately sprinkled on top before cells dried up and the dish was tapped ~10 times against the bench. This tapping causes the beads to roll over cells and induce small tears into which the Fab can diffuse in. Fourth, the stored medium was quickly added back to the cells, again to prevent cells from drying out. Cells were then placed in the incubator to recover for 1–2 h. Post-recovery, the glass beads were gently washed out with phenol red-free DMEM (DMEM⁻, Thermo Fisher Scientific, 31053-028), and the cells were stored in DMEM⁺ medium (DMEM⁻ supplemented with 10% FBS, 10 U/mL P/S, and 1 mM l-glut) for live-imaging experiments.