RNA immunoprecipitation (RIP)

Cells were grown to logarithmic phase (OD₆₀₀ = 1.0), harvested, and lysed with glass beads in 200 μl FA lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.4 U/μl RNasin, 1X protease inhibitor cocktail). Immunoprecipitation was carried out using an anti-PAP antibody (Sigma-Aldrich, St. Louis, MO, USA) and protein G-sepharose bead (GE healthcare, Chicago, IL, USA). Immunopellets were washed three times and suspended in ChIP elution buffer (100 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% SDS, 0.4 U/μl RNasin). The 5M NaCl and 0.1 μg/μl proteinase were added to 150μl of supernatant and the mixture was incubated at 42°C for 1 hr, and then at 65°C for 1 hr. RNA preparation was carried out as described above and RT-PCR were carried out using STE12-specific or ASH1-specific primers (Table 2).