GoldCLIP: Gel-omitted Ligation-dependent CLIP

研究背景

RNA结合蛋白 (RNA binding protein，RBP) 在调节RNA的定位、结构以及代谢等方面具有重要的作用，RNA结合蛋白功能的研究主要使用的方法是交联免疫沉淀技术 (Cross-Linking Immuno-Precipitation，CLIP)，然而传统的CLIP技术依赖于同位素³²P标记示踪RNA后进行转膜纯化RNA-P32/RBP复合物，极大地限制了CLIP技术的广泛使用。Promega公司研发的Halo标签因其能够与配体共价结合，具有极高的配体亲和力，为突破传统CLIP技术的局限提供了可能。

材料与试剂

1. 1.5 ml Eppendorf LoBind (Eppendorf, catalog number: 0030108116)
   
2. 2 ml Eppendorf LoBind (Eppendorf, catalog number: 0030108132)
   
3. 0.22 µm filter(Millipore，Catalog number: SLGV033RS)
   
4. Protease inhibitor cocktail (Promega, catalog number: G6521)
   
5. Micrococal nuclease (NEB, catalog number: M0247S)
   
6. Calf intestine phosphatase (NEB, catalog number: M0290S)
   
7. DNase I (Promega, catalog number: M6101)
   
8. RNase inhibitor (Promega, catalog number: N261B)
   
9. T4 RNA Ligase I (Ambion, catalog number: AM2141)
   
10. Trizol LS reagent (Invitrogen, catalog number: 10296028)
    
11. ATP (NEB, catalog number: P0756L)
    
12. DMSO (Sigma, catalog number: D8418)
    
13. Saturated phenol (Sigma, catalog number: P1037)
    
14. Glycogen (Ambion, catalog number: AM9510)
    
15. 3 M NaOAc pH 5.5 (Ambion, catalog number: AM9740)
    
16. Protease K (NEB, catalog number: P8102S)
    
17. SuperScript III reverse transcriptase (Invitrogen, catalog number: 18080093)
    
18. Halo Magne^® Beads (Promega, catalog number: G7281)
    
19. CircLigase II (Epicentre, catalog number: CK9025K)
    
20. 1 M Tris-HCl (Invitrogen, catalog number: 15568025)
    
21. 5 M NaCl (Invitrogen, catalog number: 24740011)
    
22. 0.1 M DTT (Thermo Scientific, catalog number: 707265ML)
    
23. CaCl₂ (Sigma-Aldrich, catalog number: 10035-04-8)
    
24. 10% Triton X-100 (Thermo Scientific, catalog number: R21902)
    
25. 50% Glycerol (Invitrogen, catalog number: 15514011)
26. EDTA (0.5 M), pH 8.0, RNase-free (Invitrogen, catalog number: AM9260G)
    
27. EGTA (Sigma-Aldrich, catalog number: 03777-10G)
    
28. Urea (Invitrogen, catalog number: 15505050)
    
29. 10%SDS (Invitrogen, catalog number: 15553027)
    
30. DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977023)
    
31. TBE-Urea Sample Buffer (Invitrogen, catalog number: LC6876)
    
32. Halo Magne^® Beads (Promega, catalog number: G7281)
    
33. NEB CutSmart Buffer (NEB, catalog number: B7204S)
    
34. Glycogen (Invitrogen, catalog number: 10814010)
    
35. Phusion Master Mix (NEB, catalog number: M0531L)
    
36. Zymoclean Gel DNA Recovery Kit (Zymo，catalog number：D4008)
    
37. Lysis Buffer (see Recipes)
    
38. Dilution Buffer (see Recipes)
    
39. PBST Wash Buffer (see Recipes)
    
40. High-salt PBST Wash Buffer (see Recipes)
    
41. Urea Wash Buffer (see Recipes)
    
42. SDS Wash Buffer (see Recipes)
    
43. PNK Buffer (see Recipes)
    
44. TEV Reaction Buffer (see Recipes)
    

仪器设备

1. 10 cm Petri dish
   
2. Cell spatula
   
3. 15 ml tube
   
4. Pipettes
   
5. UVLink 1000 Crosslinker (Analytik Jena)
   
6. Centrifuge (Eppendorf, model: 5424R)
   
7. Thermomixer (Eppendorf, catalog number: 5382000023)
   

实验步骤