Materials and Reagents

Note: Catalog numbers are given for most of the reagents listed below; an equivalent quality reagent from an alternative supplier can typically be substituted with comparable results. Due to the potential for artifacts introduced by contaminating RNases, care should be taken to follow best practices, such as the use of RNase-free solutions and reagents and/or using DEPC-treatment where appropriate (Farrell, 2010). Standard materials and reagents for urea-polyacrylamide gel electrophoresis are required; we use the National Diagnostics system but such gels can be prepared using standard methods (Sambrook and Russell, 2006).

1. Pipette tips 
   
2. 1.5 ml microcentrifuge tubes (e.g., Eppendorf, catalog number: 022363204 )
   
3. 10-well Gel Combs, 1.5 mm (Thermo Fisher Scientific, Novex^TM, catalog number: NC3510 )
   
4. Empty Gel Cassettes, mini, 1.5 mm (Thermo Fisher Scientific, Novex^TM, catalog number: NC2015 )
   
5. Syringe with a bent needle (to wash the residual urea out of the gel wells before sample loading)
   
6. HEK-293 Flp-In T-REx EXOSC10-3xFLAG cells ([Domanski et al., 2016]; available upon request) Parental HEK-293 Flp-In T-REx cell line (Thermo Fisher Scientific, Invitrogen^TM, catalog number: R78007 )
   
7. Nuclease-free water (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9932 )
   
8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
   
9. 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457 )
   
10. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    
11. Anti-FLAG M2 (Sigma-Aldrich, catalog number: F3165 ) antibody conjugated Dynabeads M-270 Epoxy (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 14302D ) (Domanski and LaCava, 2017)
    
12. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
    
13. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
    
14. RNA substrate with 6-carboxyfluorescein (6-FAM) at the 5’-end (Reconstitute in 10 mM HEPES ~pH 7-7.5, at 0.5 nmol/µl. Store aliquots at -80 °C)
    
    1. Generic substrate: 5’-(6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2’-O-Me)-3’
       
    2. Blocked substrate: 5’-(6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2’-O-Me, 3’-PO₄)-3’
    Note: Both substrates were ordered from the Integrated DNA Technologies at 100 nmole scale, purified by RNase-free HPLC.
    
15. RNasin^® ribonuclease inhibitors (Promega, catalog number: N2515 )
    
16. Formamide (Sigma-Aldrich, catalog number: 47671 )
    
17. DNA loading dye (Thermo Fisher Scientific, catalog number: R0611 )
    Note: This consists of 10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA–and so, can be prepared rather than purchased.
    
18. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
    
19. Tris base (Sigma-Aldrich, catalog number: 93362 )
    
20. Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: B7901 )
    
21. N,N,N’,N’-tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T7024 )
    
22. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678 )
    Note: Prepare 10% solution in H₂O and store at 4 °C for several weeks.
    
23. UreaGel 29:1 Denaturing Gel System (National Diagnostics, catalog number: EC-829 )
    
24. Recapture solution (see Recipes)
    
25. Wash solution (see Recipes)
    
26. 2x reaction solution (see Recipes)
    
27. 2x RNA loading solution (see Recipes)
    
28. 1x TBE (5x or 10x stock can be prepared) (see Recipes)
    

Equipment

Note: Standard equipment for urea-polyacrylamide gel electrophoresis is required, as well as an imager capable of fluorescein detection (absorption λ_max = 494 nm, emission λ_max = 518 nm).

1. Pipettes 
   
2. Vortexer
   
3. Benchtop mini-centrifuge
   
4. Neodymium magnet microfuge tube rack (Thermo Fisher Scientific, catalog number: 12321D )
   
5. Thermomixer (e.g., Eppendorf, model: ThermoMixer^® F , catalog number: 5355000.011; or equivalent)
   
6. XCell SureLock Mini (Thermo Fisher Scientific, model: SureLock^® Mini-Cell , catalog number: EI0001)
   
7. Electrophoresis power supply
   
8. Imager with blue light (460 nm) epi-illumination and a Y515-Di (long-pass) filter–i.e., SYBR green settings. E.g., Fujifilm LAS-3000 series or newer (Fujifilm, model: LAS-3000 Series ; or equivalent)
   

Procedure