Whole Transcriptome Target Sequencing: A Tail Counting Method

Nascent RNA cleavage and polyadenylation, which add polyA tails to the 3’ end of RNA, are essential steps for maturation of most protein-coding and non-coding transcripts.  As such, transcriptome analysis often starts with oligo(dT) priming for reverse transcription to convert RNA to cDNA molecules for sequencing.  Therefore, sequencing reads associated with polyA sites/regions can serve as signatures for global profiling of gene expression and examination of alternative polyadenylation.  Alternative polyadenylation at different polyA sites can yield RNA transcripts with different 3’ UTRs (untranslated regions), resulting in qualitative, quantitative and epigenetic effects on gene functions.  Up to date, several methods have been well developed to target 3’-ends using the Illumina sequencing platforms. Here we report our whole transcriptome target sequencing method developed to use the Ion Torrent™ PGM & Proton sequencing platform.  In brief, our process involves total RNA extraction, fragmentation, reverse transcription, first PCR amplification, fragment size selection, second PCR amplification and next generation sequencing for production of reads.  Overall, our procedures are similar to those developed for PAS (polyA site)-seq analysis, but we started with fragmentation of total RNA, performed amplification using the Ion Torrent™ PGM & Proton sequencing primers and carried out the antisense sequencing for direct profiling of polyA sites.  Our first library obtained over 30 million reads, which allowed us to identify over 26,000 clustered polyA sites for X. tropicalis as a model organism.