2.2 Solutions
1. 100 μM RNA probe sequences (see Note 1).
2. 100 μM Scrambled RNA probe sequence as control.
3. Ribonuclease (RNase)-free water.
4. 3 M NaOAc.
5. 0.1 M NaOAc.
6. 5 mM Sodium m-periodate in 0.1 M NaOAc (see Note 2).
7. 100 mM ATP.
8. 0.25 μg/μL Yeast tRNA in BC100 (see Subheading 2.3, item 3).
9. 6 ng/μL Modified trypsin, sequencing grade (see Note 3).
10. 13% Polyvinyl alcohol (PVA) (see Note 4).
11. 1Â TE: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0.
12. 0.02Â TE: TE diluted 1:50 with RNase-free water.
13. 100 mM KCl.
14. 200 mM KCl.
15. 80 mM MgCl 2 .
16. 6 mM MgCl 2 .
17. 2 M NaCl.
18. 100 mM NaCl.
19. 400 mM NaCl.
20. 0.5 M Creatine phosphate.
21. Adipic acid dihydrazide agarose beads in 50% solution (see
Note 5).
22. 20 mM Tris–HCl, pH 7.5.
23. 100% Ethanol.
24. 80% Ethanol.
25. 5 mg/mL Glycogen.
26. 100 mM NH 4 CO 3 .
27. Protein lysate (45–50 μg per reaction; see Subheading 3.2).
2.3 Buffers
1. GFB100: 0.02Â TE, 100 mM KCl.
2. GFB200+ 6 mM MgCl 2 : 0.02Â TE, 200 mM KCl, 6 mM
MgCl 2 .
3. BC100: 0.02Â TE, 100 mM NaCl.
4. BC400: 0.02Â TE, 400 mM NaCl.
5. Binding buffer (per reaction): 397.5 μL BC100, 316.5 μL
RNase-free water, 150 μL BC400, 150 μL 13% PVA, 150 μL
10 mM ATP, 150 μL 0.25 μg/μL tRNA in BC100, 60 μL
80 mM MgCl 2, 60 μL 0.5 M creatine phosphate, 45–50 μg
protein lysate (no more than 60 μL).
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1. 100 μM RNA probe sequences (see Note 1).
2. 100 μM Scrambled RNA probe sequence as control.
3. Ribonuclease (RNase)-free water.
4. 3 M NaOAc.
5. 0.1 M NaOAc.
6. 5 mM Sodium m-periodate in 0.1 M NaOAc (see Note 2).
7. 100 mM ATP.
8. 0.25 μg/μL Yeast tRNA in BC100 (see Subheading 2.3, item 3).
9. 6 ng/μL Modified trypsin, sequencing grade (see Note 3).
10. 13% Polyvinyl alcohol (PVA) (see Note 4).
11. 1Â TE: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0.
12. 0.02Â TE: TE diluted 1:50 with RNase-free water.
13. 100 mM KCl.
14. 200 mM KCl.
15. 80 mM MgCl 2 .
16. 6 mM MgCl 2 .
17. 2 M NaCl.
18. 100 mM NaCl.
19. 400 mM NaCl.
20. 0.5 M Creatine phosphate.
21. Adipic acid dihydrazide agarose beads in 50% solution (see
Note 5).
22. 20 mM Tris–HCl, pH 7.5.
23. 100% Ethanol.
24. 80% Ethanol.
25. 5 mg/mL Glycogen.
26. 100 mM NH 4 CO 3 .
27. Protein lysate (45–50 μg per reaction; see Subheading 3.2).
2.3 Buffers
1. GFB100: 0.02Â TE, 100 mM KCl.
2. GFB200+ 6 mM MgCl 2 : 0.02Â TE, 200 mM KCl, 6 mM
MgCl 2 .
3. BC100: 0.02Â TE, 100 mM NaCl.
4. BC400: 0.02Â TE, 400 mM NaCl.
5. Binding buffer (per reaction): 397.5 μL BC100, 316.5 μL
RNase-free water, 150 μL BC400, 150 μL 13% PVA, 150 μL
10 mM ATP, 150 μL 0.25 μg/μL tRNA in BC100, 60 μL
80 mM MgCl 2, 60 μL 0.5 M creatine phosphate, 45–50 μg
protein lysate (no more than 60 μL).
244
Mengge Shan and Brian D. Gregory