RIP-Seq

RIP-Seq

RNA immunoprecipitation sequencing (RIP-Seq) maps the sites where proteins are bound to the RNA within RNA-protein complexes . In this method, RNA-protein complexes are immunoprecipitated with antibodies targeted to the protein of interest. After RNase digestion, RNA covered by protein is extracted and reverse-transcribed to cDNA. The locations can then be mapped back to the genome. Deep sequencing of cDNA provides single-base resolution of bound RNA.

Pros:

• Maps specific protein0RNA complexes, such as polycomb-associated RNAs
• Low background and higher resolution of binding site due to RNase digestion
• No prior knowledge of the RNA is required
• Genome-wide RNA screen

Cons:

• Requires antibodies to the targeted proteins
• Nonspecific antibodies will precipitate nonspecific complexes
• Lack of crosslinking or stabilization of the complexes may lead to false negatives
• RNase digestion must be carefully controlled

• RIP-Seq: Zhao J., Ohsumi T. K., Kung J. T., Ogawa Y., Grau D. J., et al. (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40: 939-953

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