Materials and Reagents

1. Sucrose
   
2. 1 M HEPES-KOH (pH 7.6)
   
3. 2 M KCl
   
4. 1 M MgCl₂
   
5. PBS
   
6. 1 M Tris-HCl (pH 7.5)
   
7. 10 mg/ml cycloheximide (Sigma-Aldrich, catalog number: C7698 ) in MilliQ water
   Note: Cycloheximide inhibits protein synthesis by blocking translation elongation. This molecule interferes with the translocation of tRNAs with the mRNA and the ribosome, resulting in fixed ribosomes on mRNAs.
8. Protease inhibitor cocktail (EDTA-free) (F. Hoffmann-La Roche, catalog number: 04 693 159 001 )
   
9. RNase inhibitor (RNasin 40 units/μl) (Promega Corporation, catalog number: N2511 )
   
10. 10% Triton X-100
    
11. 10% Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
    
12. RNaseZap (Life Technologies, Ambion^®, catalog number: AM9780 )
    
13. Trizol (Life Technologies, Invitrogen^TM)
    
14. Oligo dT primer and Super Script III (Life Technologies, Invitrogen^TM, catalog number: 18418012 and 18080093 )
    
15. 10x sucrose gradient buffer (see Recipes)
    
16. 10-50% sucrose solutions (see Recipes)
    
17. Hypotonic buffer (see Recipes)
    

Equipment

1. Ultra centrifuge and rotor (Beckman, model: Optima L-80 ; SW40Ti rotor )
   
2. UV detector and fraction collector (Teledyne ISCO)
   
3. NanoDrop (Thermo Fisher Scientific)
4. 0.22 µm filter
   

Software

1. TracerDAQ software (MicroDAQ)

Procedure