Polyribosome Profiling

Materials and Reagents

1. Acetone
2. Sucrose
3. Cyclohexamide
4. Bromophenol blue (BPB)
5. Urea
6. Tris-HCl
7. Tris-Acetate
8. MgCl₂
9. DTT
10. NH₄Cl
11. NaCl
12. DEPC-treated water
13. Heparin
14. Stocks (see Recipes)
15. Lysis buffer (see Recipes)
16. 60% sucrose (see Recipes)
17. 7% sucrose solution (see Recipes)
18. 47% sucrose solution (see Recipes)
19. Diluted sucrose (see Recipes)
20. Cyclohexamide to add to cells (see Recipes)
    

Equipment

1. Autoclaved, RNase free 500 ml centrifuge bottles (1 per sample)
2. Autoclaved, RNase free 40 ml centrifuge bottles (1 per sample)
3. Centrifuges
4. Colored Epi tubes. Need 30/sample
5. 15 ml, 50 ml falcon tubes (BD Biosciences, Falcon^®)
6. Ultracentrifuge tubes (Don't autoclave) (Beckman Coulter, catalog number: 344059 )
7. Beckman JA-10, JA-20, and Epi rotors to 4 °C (Beckman Coulter)
8. Ultra SW-51 buckets to 4 °C
9. RNase-free glass beads and bead scoop
10. Foxy fraction collector
11. BioRad Econo UV monitor (Bio-Rad)
12. Peristaltic pump
    

Procedure