Here, scientists at Academia Sinica, Taiwan 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.
- 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.