Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform

The 5′ untranslated regions of mRNA play an important role in their translation.Here, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript) technology, while the second involves replacing the 5′ cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5′ end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5′ end.These methods are suitable for global mapping of mRNA 5′ ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure