Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth Lyon; Luis U. Aguilera; Tatsuya Morisaki; Brian Munsky; Timothy J. Stasevich

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/478040 doi: bioRxiv preprint sfGFP and 33 µg/ml of purified MCP-HaloTag protein were mixed with PBS to a final volume of 4 µl. Second, in cell culture hood, DMEM was aspirated from the MatTek chamber and the 4 µl mix was pipetted on top of cells. Third, ~106 µm glass beads (Sigma Aldrich) were evenly sprinkled over the cells. Fourth, the chamber was tapped carefully ~10 times on the cell culture hood bench top. Fifth, DMEM was immediately added back to the cells. Sixth, cells were returned to the incubator for at least an hour to recover from the loading procedure. In most experiments, we also bead-loaded DNA, which we added to the initial 4 µl mix (so that DNA had a final concentration close to 1 mg/ml). On occasion, DNA was transiently transfected ~2 hours before bead-loading. Around one hour before experiments began, bead-loaded cells were washed with phenol-red-free complete DMEM to remove glass beads, and 200 nM of JF646-HaloTag ligand (a cell permeable fluorogenic ligand (Grimm et al., 2015) ) was added to label MCP-HaloTag protein. After 30 mins of incubation, cells were washed three times using phenol-red-free complete DMEM to remove any unconjugated fluorophores. Cells were then immediately imaged for experiments.