Ti eclipse wide field microscope

All phase separation assays were performed by cleaving the 6x-His and MBP tags from purified FUS proteins with TEV protease as in previous studies (Rhine et al., 2020a (link); Rhine et al., 2021 ). Successful cleavage of all proteins was verified with SDS-PAGE and Coomassie staining (data not shown). FUS protein was buffer-exchanged from the elution buffer into 20 mM Na₃PO₄, pH 8.0, using successive spins in Amicon filters. In general, PAR phase separation reactions used FUS (1 μM), unlabeled PAR (90 nM), and Cy5-labeled PAR (10 nM) in 1X Cleavage Buffer (100 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, and 1 mM EDTA, pH 8), unless otherwise indicated. RNA droplet reactions generally used 1 μM RNA and 10 nM Cy3-RNA. For conditions with 10 nM or less PAR, all PAR was Cy5-labeled and there was no unlabeled PAR. Reactions (200 μL) were incubated at room temperature for 4 h in Nunc Lab-Tek 8-well chambers. Images were acquired with a Nikon Ti Eclipse wide-field microscope in the brightfield, Cy3, and/or Cy5 channels.

A Nikon Ti Eclipse widefield microscope was used to visualize DNA using DAPI (405 nm), GFP-tubulin or secondary antibody on immunostained tubulin (488 nm), and TagRFP-T (561 nm). Cells were scored as misaligned if their chromosomes were more than 50% of the distance between the spindle pole and the metaphase plate. As is common with S2 cell cultures, ~50% of the cells do not express the protein of interest, and provide an internal control for our experiments.