CLIP-seq Protocol for Identifying in vivo Targets of RNA Binding Proteins

材料与试剂

1. 15 cm培养皿
2. 15 ml离心管
3. 移液器枪头
4. DNA/RNA lobind tube 1.5 ml (Eppendorf, catalog number: 022431021)
5. DNA/RNA lobind tube 2.0 ml (Eppendorf, catalog number: 022431048)
6. HeLa细胞
7. RQI RNase-free DNase (Promega, catalog number: M6101)
8. DDX17 antibody (Proteintech, catalog number: 19910-1-AP)
9. Normal Rabbit IgG (CST, catalog number: 2729)
10. RNAsin (Promega, catalog number: N2611)
11. Micrococcal Nuclease (Thermo Scientific, catalog number: EN0181)
12. Dynabeads Protein G (Life Technologies, catalog number: 10004D)
13. FastAP (Life Technologies, catalog number: EF0652)
14. T4 RNA ligase 2 tr KQ (NEB, catalog number: M0351)
15. T4 PNK (NEB, catalog number: M0201L)
16. T4 RNA ligase 1 high concentration (NEB, catalog number: M0437M)
17. ATP (Invitrogen, catalog number: 18330019)
18. 32P-γ-ATP (PerkinElmer, catalog number: NEG502A250UC)
19. NuPAGE 4%~12% Bis-Tris Gel (Invitrogen, catalog number: NP0321BOX)
20. NuPAGE LDS Sample Buffer (Invitrogen, catalog number: NP0007)
21. NuPAGE MOPS SDS Running Buffer (20×) (Invitrogen, catalog number: NP0001)
22. NuPAGE Transfer Buffer (20×) (Invitrogen, catalog number: NP0006)
23. Proteinase K (Thermo Scientific, catalog number: EO0491)
24. Sodium Acetate Solution (3 M), pH 5.2 (Thermo Scientific, catalog number: R1181)
25. GlycoBlue Coprecipitant (Invitrogen, catalog number: AM9515)
26. SuperScript III Reverse Transcriptase (Invitrogen, catalog number: 18080044)
27. Exo-SAP-IT (Affymetrix, catalog number: 78201)
28. Dynabeads MyOne Silane (Life Technologies, catalog number: 37002D)
29. RLT buffer (Qiagen, catalog number: 79216)
30. Phusion High-Fidelity DNA polymerase (Thermo Scientific, catalog number: F530S)
31. QIAEX II Gel Extraction Kit (Qiagen, catalog number: 20021)
32. Oligos (Takara)
    3’ APP-DNA linker：
    rAppp-TGTAGGCACCATCAAT-NH2
    5’ DNA linker：
    5Phos/NNNNNNAGATCGGAAGAGCGTCGTGT/3spc3
    RT primer：
    TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG
    short xrn1 F primer：
    ACACGACGCTCTTCCGATCT
    short xrn1 R primer：
    TTCAGACGTGTGCTCTTCCGATCT
    long xrn1 F primer：
    AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    A701R primer：
    CAAGCAGAAGACGGCATACGAGATGTCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
    A702R primer：
    CAAGCAGAAGACGGCATACGAGATACCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
33. SDS (Biosharp, catalog number: BS088)
34. NP-40 (Amresco, catalog number: M158)
35. 1M Tris-HCl (pH 7.5) (Invitrogen, catalog number: 15567-027)
36. MgCl2·6H₂O (上海国药, catalog number: 10012818)
37. EGTA (Millipore, catalog number: 324626)
38. NaOH (上海国药, catalog number: 10019718)
39. NaCl (上海国药, catalog number: 10019318)
40. Na₂EDTA (Sigma, catalog number: E5134)
41. MOPS (Sigma, catalog number: M1254)
42. Tris (Amresco, catalog number: 0497)
43. Bicine (Sigma, catalog number: B3876)
44. Bis-Tris (Sigma, catalog number: B9754)
45. 甲醇 (上海国药, catalog number: 10014118)
46. PEG 8000 (US Pharmacopeia, catalog number: 1546605)
47. 3M醋酸钠 (pH 5.5) (Ambion, catalog number: AM9740)
48. Glycoblue (Ambion, catalog number: AM9515)
49. 无水乙醇 (上海国药, catalog number: 10009218)
50. 异丙醇 (上海国药, catalog number: 80109218)
51. 氯仿 (上海国药, catalog number: 10006818)
52. 水饱和酚 (Ambion, catalog number: AM9720)
53. Washing buffer (见溶液配方)
54. PNK buffer (见溶液配方)
55. PNK + EGTA buffer (见溶液配方)
56. MN buffer (见溶液配方)
57. PK buffer (见溶液配方)
58. 20× MOPS buffer (见溶液配方)
59. 20× Transfer buffer (见溶液配方)
60. 1× Transfer buffer (见溶液配方)
    

仪器设备

1. 移液器
2. -80 °C冰箱
3. 普通PCR仪 (Bio-Rad)
4. 普通离心机及冷冻离心机 (Eppendorf)
5. 热混仪 (Eppendorf)
6. 紫外交联仪 (UVP HL-2000 Hybrilinker)
7. 紫外凝胶成像系统 (Gene Genius)
8. 电泳仪、转膜槽 (北京六一公司)
9. 跑胶槽XCell SureLock Mini-Cell (Invitrogen)
10. 超净工作台 (哈东联公司)
11. 生物安全柜 (Heal Force)
12. 细胞培养箱(Thermo scientific)
13. pH计 (梅特勒-托利delta-320)
14. 超低温冰箱 (三洋)
15. 暗盒、X胶片 (富士)
16. 自动洗片机 (江苏泰星)
17. 盖革计数器 (model3美国Iudlum公司)
    

实验步骤