A 485

To generate acetylated nucleosome arrays for microinjection, recombinant p300 histone acetyl transferase domain was generated (VBCF Protein technologies Facility) using plasmid pET duet+p300_HAT according to a purification strategy described previously^{13 (link)}. To induce histone acetylation of nucleosome arrays, arrays with 8% Alexa Fluor 488 or Alexa Fluor 594 label density at a concentration of 3.85 μM were incubated with 6.12 μM p300-HAT (enzyme stock 61.2 μM in gel-filtration buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 10% (w/v) glycerol, 1 mM DTT)) in the presence of 750 μM Acetyl-CoA (Sigma-Aldrich, A2056) in gel-filtration buffer at room temperature for 2 h with occasional agitation. The acetylation was next stopped by the addition of A-485 (Tocris, 6387) to a final concentration of 9 μM and the reaction mixture was stored in the dark. To 10 μl of quenched acetylation reaction, 10 μl dilution buffer containing 5 μM A-485 (Tocris, 6387) was added and the reaction was allowed to equilibrate at room temperature for 10 min. Next, 1 volume of phase-separation buffer containing 5 μM A-485 (Tocris, 6387) was added and the mixture was incubated for 10 min at room temperature before transferring the suspension to a well of a passivated 384-well microscopy plate.