Figure 5

Both RNase inhibitors are needed to keep the RNA intact when cells are lysed by mortar grinding. A, Strain RJY1885 (She2p-TAP) was ground in a motor-driven mortar in the presence of dry ice. Samples from crude lysate and supernatants of 1200 × g (S1) and 7400 × g (S7) spins and pellet from 7400 × g spin (P7), as well as input material (IgG input) and flow through (IgG FT) from the IgG immunopurification, were phenol:chloroform extracted as indicated in Methods. 8 μg from the extracted RNA were loaded onto 1.2% agarose-formaldehyde gels. As RNA quality control, 8 μg of total RNA (P/C lysis RNA) extracted from the same strain with a phenol extraction method [18] were loaded in parallel. Membranes were first probed against ASH1 mRNA, stripped and then probed against PDA1 mRNA. The positions of the 25S and 18S ribosomal RNAs are shown, as well as the PDA1 and ASH1 mRNA hybridization signals. B, Sub2p-TAP strain [24] was ground in a motor-driven mortar in the presence of dry ice and thawed in lysis buffer supplemented with 50 U/ml of SuperaseIn and/or 5 mM RVC. Samples from crude lysate and supernatants of 100000 × g (S100) spin and flow through (IgG FT) from the IgG immunopurification, were phenol:chloroform extracted as indicated in Methods. 8 μg from the extracted RNA were loaded onto 1.2% agarose-formaldehyde gels. As RNA quality control, 8 μg of total RNA (P/C lysis RNA) extracted from the same strain with a phenol extraction method [18] were loaded in parallel. The positions of the 25S and 18S ribosomal RNAs are shown, as well as the PDA1 mRNA hybridization signal.