Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5′-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing

Abstract

This protocol describes purification of RNA cross-linking immunoprecipitation (CLIP) tags by proteinase K digestion of the cross-linked protein, addition of a 5′ linker to the RNA tags, and amplification of the product by transcription-polymerase chain reaction (RT-PCR). Use of this protocol adds another important purification step: sizing of the PCR products to enrich for those derived from RNA originally cross-linked to the desired RNABP. Finally, sequencing of the PCR products is described. There are two strategies for sequencing the PCR products of “CLIPed” RNA. Low-throughput sequencing involves cloning of PCR products, conventional minipreps, and sequencing. This can be performed on the PCR products generated here using standard protocols for A-tailing the PCR product and TA-cloning. This may be a worthwhile strategy when analyzing a small number of clones. In general, particularly in light of falling costs, high-throughput sequencing is the preferred method for sequencing the products of CLIPed RNA. This protocol describes a method for reamplifying PCR products with primers suitable for use on Illumina’s Solexa platform. Although this protocol is specific to the Illumina deep-sequencing platform, similar schemes for reamplification of the initial PCR products can be used to add platform-specific sequences to the termini of the PCR-amplified DNA.