實驗概要
Interest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs. CLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP), but differs from RIP in the use of UV radiation to cross-link RNA binding proteins to the RNA that they are bound to. This covalent bond is irreversible, allowing stringent purification conditions. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA. Different types of CLIP exist, high-throughput sequencing-CLIP (HITS-CLIP), Photoactivatable-Ribonucleoside Enhanced CLIP (PAR-CLIP), and Individual CLIP (iCLIP). Here is a summary of the iCLIP protocol adapted from Konig et al. J. Vis. Exp. 2011.
主要試劑
1. Lysis buffer
50 mM Tris-HCl, pH 7.4
100 mM NaCl
1% NP-40
0.1% SDS
0.5% sodium deoxycholate
Protease inhibitors (add fresh each time)
2. High-salt buffer
50 mM Tris-HCl, pH 7.4
1 M NaCl
1 mM EDTA
1% NP-40
0.1% SDS
0.5% sodium deoxycholate
3. Low RNase dilution
1/500 RNase I dilutions for library preparation
High RNase dilution
1/50 RNase I dilutions to control for antibody specificity
4. Wash buffer
20 mM Tris-HCl, pH 7.4
10 mM MgCl2
0.2% Tween-20,/p>
5. PNK mix
15 μl water
4 μl 5x PNK pH 6.5 buffer [350 mM Tris-HCl, pH 6.5; 50 mM MgCl2; 25 mM dithiothreitol];
0.5 μl PNK enzyme
0.5 μl RNasin
6. Ligation mix A
9 μl water
4 μl 4x ligation buffer [200 mM Tris-HCl; 40 mM MgCl2; 40 mM dithiothreitol]
1 μl RNA ligase
0.5 μl RNasin
1.5 μl pre-adenylated linker L3 [20 μM]
4 μl PEG400
7. Hot PNK mix
0.4 μl PNK
0.8 μl 32P-γ-ATP
0.8 μl 10x PNK buffer
6 μl water
8. PK buffer
100 mM Tris-HCl pH 7.4
50 mM NaCl
10 mM EDTA
9. PKurea buffer
100 mM Tris-HCl pH 7.4
50 mM NaCl
10 mM EDTA
7 M urea
10. RNA/primer mix
6. 25 μl water
0.5 μl Rclip primer [0.5 pmol/μl]
0.5 μl dNTP mix [10 mM]
11. Oligo annealing mix
26 μl water
3 μl FastDigest Buffer
1 μl cut oligo [10 μM]
12. Ligation mix B
6.5 μl water
0.8 μl 10x CircLigase Buffer II
0.4 μl 50 mM MnCl2
0.3 μl Circligase II
13. Oligo annealing mix
26 μl water
3 μl FastDigest Buffer
1 μl cut oligo [10 μM]
14. PCR mix
19 μl cDNA
1 μl primer mix P5/P3 solexa
10 μM each
20 μl Accuprime Supermix 1 enzyme