Anti ha affinity resin

Cells expressing an HA-tagged version of Csr2 (endogenously tagged at its genomic locus; ySL1037) were grown overnight at 30°C in glucose-containing SC medium to an A₆₀₀ of 0.3–0.6 and transferred to SC-lactate medium for 4 h at 30°C. Cells from half of the culture (∼400 OD units) were collected by centrifugation (5 min, 4,000 g, 4°C). Glucose (2% final) was added to the other half, and cells were incubated for 5 min at 30°C before being harvested. Each pellet was resuspended in 4 ml lysis buffer (200 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM DTT, and 0.1% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [Sigma-Aldrich], 10 mM N-ethylmaleimide [Sigma-Aldrich], 100 µM MG-132 [Enzo Life Sciences], and 1% yeast protease inhibitor cocktail [P8215; Sigma-Aldrich]). Cells were mechanically disrupted with glass beads on a vortex at 4°C for 4 × 30 s. The lysate was then cleared at 3,000 g for 5 min, at 4°C, and the supernatant was incubated with 25 µl anti-HA affinity resin (Roche) for 1 h 30 min at 4°C under rotation. The beads were then washed three times with lysis buffer and once with H₂O and resuspended in 100 µl H₂O, before being subjected to trypsin digestion and mass spectrometry analysis.

Plasmids containing DCL3 and DCL4 Flag-HA were linearized and injected into the macronucleus of vegetative cells. 1.2x10⁶ of cells (400 mL culture) were harvested at an early developmental stage and cell pellets were frozen in liquid nitrogen. Immunoprecipitation was performed as described in Reuter et al. (2009) (link) and Cora et al., (2014) (link). In detail, the cell pellets were resuspended in 2 mL fresh lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.5% sodium deoxycholate, 1% Triton X-100, 1X protease inhibitor complete tablet (Roche), and 10% glycerol) and homogenized until complete lysis. The cell lysates were centrifuged at 13000x g for 15 min at 4°C. 1 mL of the supernatant was incubated with 50 μL of Anti-HA affinity resin (Roche) overnight at 4°C while rotating. Beads were washed three times with 1 mL IP buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.01% NP-40, 1 mM MgCl₂ 1X protease inhibitor and 5% glycerol) before incubation. After overnight incubation, beads were washed five times with 1 mL IP buffer. Washed beads were resuspended in 100 μL IP buffer and aliquots of 20 μL were stored at −20°C until further use.