Total RNA From Yeast Using Glass Beads

Total RNA From Yeast Using Glass Beads
In advance, prepare 2 ml tubes containing 0.25 g acid washed glass beads (0.5 mm diameter) and 250 µl phenol:CHCl3:isoamyl alcohol (25:24:1). Label and place on ice.
1. Collect 5 OD600 units of yeast (e.g., 10 mls of OD600 = 0.5). Centrifuge for 5 minutes at 2000 rpm
at 4°C. Resuspend pellet in 2 mls of ice cold HE buffer and transfer to 2 microfuge tubes. Spin for 30 seconds at top speed at 4°C. Keep pellets on ice for immediate use or store at -80°C.
2. Resuspend cell pellet in 250 µl HENS buffer. Quickly transfer to 2 ml tube with glass beads.
Close tube quickly but carefully (glass beads can interfere with cap closure). Vortex for 10
seconds. Place tube on ice. Do one tube at a time.
3. Parafilm tubes to secure tops. Vortex in multi-tube mixer at 4°C for 30 minutes.
4. Freeze in powdered dry ice for 10 minutes (or longer).
5. Microfuge for 5 minutes at top speed at 25°C. The tube will thaw and an interface layer will
separate the upper aqueous and lower phenol layers. If the interface is broad, spin longer at 4°C.
6. Transfer ~200 µl of aqueous supernatant to a 1.5 ml microfuge tube. Do not collect any interface.
7. Repeat extraction of aqeuous layer. Add equal volume of phenol:CHCl3:IAA. Vortex 15 seconds.
Spin 5 minutes. Transfer supernatant to fresh 1.5 ml tube.
8. Add 3 volumes of 100% EtOH to supernatant. Precipitate 1 hour to overnight at -20°C. Do not
add additional salt (e.g., don’t add NaOAc, pH 5). Optional: DEPC treat EtOH.
9. Spin for 30 minutes at top speed at 4°C. Carefully remove supernatant. Pellet may be small.
10. Wash pellet with of 75% EtOH (prepared with DEPC treated dH2O). Spin for 5 minutes at top
speed. Carefully remove supernatant. Spin briefly and remove droplets.
11. Resuspend pellet in DEPC-treated dH2O (10 µl per 1 OD600 unit). Alternatively, resuspend RNA
in dH2O + 0.1 % SDS + 0.1 mg/ml proteinase K. Keep RNA on ice. Store RNA at -80°C.
HE Buffer: HENS Buffer:
10 mM HEPES-NaOH, pH 7.5 10 mM HEPES-NaOH, pH 7.5
1 mM EDTA 1 mM EDTA
300 mM NaCl
0.2% SDS
Treat HE, HENS, and dH2O with DEPC – add 0.1% DEPC, hold 1 hour at 37°C, autoclave.
For pulse-chase experiments. At step 1: Start with 0.5 OD600 unit of labeled yeast cells per time point. Collect cells from different time points by centrifugation and store at -80°C. At step 6: collect exactly the same volume of supernatant from each tube. For small amounts of cells, back extract the phenol layer with 100 µl of HENS buffer and pool with 200 µl.。