6857 freezer mill

The C-terminus of the Spt20 subunit of the SAGA complex was tagged with a TAP tag. The SAGA complex was purified using a standard TAP procedure as described previously^{46 (link)}. Typically, 10 L of yeast cells were harvested and broken by SPEX 6857 Freezer Mill (SPEXSamplePrep, USA). After centrifugation at 200,000 g for 1 h at 4 °C, the supernatant was incubated with 800 μL prewashed IgG-Sepharose beads (GE Healthcare, 17096901, USA) for 4 h at 4 °C. The protein sample was released from the beads by TEV protease digestion for 4 h. The eluted protein complex was concentrated to 40–50 μL for in vitro acetylation and benzoylation assays.

The C terminus of the Sth1 subunit of RSC was tagged with a TAP tag. The RSC complex was purified using a standard TAP procedure as described before^{34 (link)}. Typically, 10 L of cells were harvested and broken by SPEX 6857 Freezer Mill (SPEXSamplePrep). The lysate was centrifuged at 200,000 × g for 1 h at 4 °C. The supernatant was incubated with IgG-Sepharose beads (GE Healthcare) for 3 h at 4 °C. The protein sample was released from the beads by TEV protease digestion for 2 h at 4 °C. The sample was then incubated with calmodulin affinity beads (GE healthcare) for 3 h. The RSC complex was eluted in the elution buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM EGTA, 2 mM DTT).