Abstract
RNA seq or Direct cDNA sequencing is an emerging revolutionary tool for whole-transcriptome analysis that provides information about the structure of transcripts and their expression levels. It is rapidly gaining momentum for transcript profiling and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and SNPs, compared to microarrays. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise preparing rRNA-depleted RNA and either (i) RNA fragmentation, 5′ and 3′ adaptor-ligation, size selection, cDNA synthesis, and multiple clean-up steps; or (ii) cDNA synthesis followed by cDNA fragmentation, end-polishing, 5′ and 3′ adaptor-ligation, size selection and multiple clean-up steps. These methods are generally time-consuming and require significant hands-on time. Here we introduce an improved, user-friendly version of the ScriptSeq™ RNA-Seq library preparation method. ScriptSeq utilizes a unique Terminal-Tagging technology that simplifies the preparation of directional, paired-end libraries from rRNA-depleted or poly(A)-enriched RNA.
ScripSeq Version 2:
Libraries are prepared in about 4 hours, in a single-tube workflow
Produces directional cDNA libraries with ∼98% strandedness, which is critical for annotation of novel genes
Can be used with both intact and degraded (eg. FFPE) RNA samples
Provides very good 5′/3′ representation of transcripts
When starting from Rib-Zero treated RNA, less than 2 % of the sequence reads from libraries map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads improves sequence depth and coverage, and increases the percentage of uniquely mapped reads.