Massively parallel sequencing of 3 prime-ends of mRNAs and small RNAs with 454 sequencer in moss Physcomitrella patens

Abstract

To understand the molecular mechanisms for pluripotent stem cell formation, we have established an experimental system with the moss Physcomitrella patens, in which a differentiated leaf cell changes to an apical stem cell simultaneously with cell cycle re-entry. It is critical to identify the factors affecting this reprogramming process. We are employing the massively parallel sequencing approach for genome-wide transcriptome analysis. Comprehensive mRNA 3 prime-end and small RNA sequencing were conducted with 454 sequencer. For mRNA 3 prime-end sequencing, we developed the method in which mRNA 3 prime-ends were successfully trapped by improved primer sets. 1.9-million for 3 prime-end sequence (per five samples) and 560,000 for small RNA sequence (per one sample) tags were obtained. Mapping of 3 prime-end sequence tags revealed the high trapping efficiency of 3 prime-ends by this method.