6 reagents selected for iCLIP (including pre-adenylated linker L3)

Individual-nucleotide resolution UV CrossLinking and ImmunoPrecipitation (iCLIP) enables the identification of protein-RNA interactions. The original CLIP protocol described by Jernej Ule et al. in 2003 (1) has evolved significantly (2, 3), and the current iteration of the iCLIP protocol requires a core set of oligonucleotides and enzymes.

iCLIP is a powerful tool to perform transcriptome wide mapping of the binding sites of an RNA-binding proteins.

Nevertheless, this method requires high level of expertise and optimised reagents to obtain optimal results. When discussing this technique (which I have to admit was relatively unclear to me) with Mark,  my colleague and Molecular Biology Manager at tebu-bio, I have realized that an attractive “iCLIP bundle” based on this protocol was possible.

Here are 6 optimized reagents, including an iCLIP preadenylated linker, that previously had to be custom ordered.

Ideal “iCLIP” bundle:

Here are Mark’s suggestions:

1. iCLIP Oligo (3 x 3 nmol; TriLink Biotechnologies, cat. nr 040O-30050-9)
2. RNASE I (EpiCentre-Illumina) (cat. nr 035N6901K)
3. T4 RNA Ligase 2 (EpiCentre Illumina, cat. nr 035LR2D1132K) – ligates the preadenylated 5´ ends of DNA or RNA are ligated to the 3´ ends of RNA
4. Proteinase K (EpiCentre Illumina, cat. nr 035MPRK092)
5. MonsterScript RT Kit (EpiCentre Illumina, cat. nr 035MS041050) –  EpiCentre’s answer to SuperScript III
6. CircLigase II ssDNA Ligase (EpiCentre-Illumina, cat. nr 035CL9021K) –  circularizes ssDNA templates having a 5´-phosphate and a 3´-hydroxyl group in the absence of a complementary sequence

These 6 reagents can be used in combination with our antibodies optimized for IP.

 Want to know more about iCLIP?

You might like to access initial publications describing the initial iCLIP protocol:

1. Ule J. et al. “CLIP Identifies Nova-Regulated RNA Networks in the Brain” (2003) Science, Vol. 302 no. 5648 pp. 1212-1215. DOI: 10.1126/science.1090095
2. Ule J. et al. “CLIP: A method for identifying protein–RNA interaction sites in living cells” (2005) Methods, Vol. 37, Issue 4, pp. 376–386. DOI: 10.1016/j.ymeth.2005.07.018.
3. Konig et al. “iCLIP – Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution” (2011) J Vis Exp., Vol. 50. pii: 2638. DOI: 10.3791/2638

Feel free to leave your comments, tips or recommendations you’d like to share with other researchers for iCLIP procedures!