Robust analysis of 5'-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites.

The structure and diversity at the 3' ends of mRNA transcripts have been extensively characterized using several tag-based techniques in eukaryotes. However, the 5' ends of mRNA transcripts are not well understood, owing to a lack of efficient experimental approaches. We developed a new gene expression profiling method, called robust analysis of 5'-transcript ends (5' RATE), to rapidly isolate the 5' ends of mRNA transcripts. After ligating RNA oligo linkers to the 5' regions of decapped mRNA, cDNA is synthesized and digested with the restriction enzyme NlaIII. Ditags are formed by ligating two individual NlaIII tags, and are then PCR-amplified, purified and sequenced using a pyrosequencing approach. The 5'-RATE procedure is simple, fast and cost-effective because the complicated steps in comparative methods such as serial analysis of gene expression (including the formation of concatemers and their subsequent cloning and sequencing) have been eliminated. The longer 5'-RATE tags (>80 bp) provide more accurate matching to reference sequences for gene annotation and allow in-depth analysis of sequence diversity at the 5' regions of mRNA transcripts. Using our procedure, a 5'-RATE library with about 180,000 end sequences can be generated within a week. We have successfully applied the 5'-RATE method to characterize the transcriptome of various plant species including maize, rice and soybean. This method can be easily adapted to other eukaryotic organisms using the detailed procedures described in this protocol.