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NuGEN's portfolio of Ovation RNA-Seq sample preparation solutions provide an end-to-end workflow for strand-specific RNA-Seq library construction using nanogram amounts of total RNA obtained from a broad range of model organisms such as mouse, rat, Drosophila and Arabidopsis. The integrated workflow (Figure 1) from input of total RNA to fully constructed library is highly reproducible, and can be completed in approximately 7 hours. Insert Dependent Adaptor Cleavage (InDA-C) technology enables targeted depletion of unwanted transcripts, and minimizes sequencing reads derived from these transcripts. RNA-Seq sample preparation solutions for model organisms are designed to minimize levels of both cytoplasmic and mitochondrial rRNA, and in the case of Arabidopsis, chloroplast rRNA as well.

Each RNA-Seq solution has 16 unique barcoded adaptors to enable multiplex sequencing to further optimize efficiencies and cost savings in transcriptome analysis. The kits provide all reagents necessary for RNA-Seq library construction, including the species- and transcript-specific InDA-C primers used for targeted depletion. An example of how InDA-C technology can be used to deplete rRNA sequencing reads in mouse is shown in Table 1.

The Ovation RNA-Seq sample preparation solutions for model organisms provide strand-specific expression analysis by incorporation of
a nucleotide analogue during second strand cDNA synthesis (see Table 1 for data using the Ovation Mouse RNA-Seq System 1-16), and subsequent ligation to a pair of double-stranded library adaptors also containing the same analogue in one strand. After ligation, the cDNA strand and adaptor containing the analogue are selectively removed (Strand Selection), leaving only one cDNA strand with both adaptor sequences attached. This product is then converted into a sequence-ready library by PCR amplification (see Figure 1).


Figure 1. Ovation RNA-Seq System workflow


Table 1: Stranded RNA-Seq with Mouse Total RNA Using rRNA InDA-C Primers

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¡¤ First Strand Random Primer Mix
¡¤ First Strand Oligo dT Primer Mix
¡¤ First Strand Primer Buffer Mix
¡¤ First Strand Buffer Mix
¡¤ First Strand Enzyme Mix
¡¤ Second Strand Buffer Mix
¡¤ Second Strand Enzyme Mix
¡¤ Second Strand Stop Buffer
¡¤ End Repair Buffer Mix
¡¤ End Repair Enzyme Mix
¡¤ End Repair Enhancer
¡¤ Ligation Buffer Mix
¡¤ Ligation Enzyme Mix
¡¤ Strand Selection Buffer Mix I
¡¤ Strand Selection Enzyme Mix I
¡¤ Strand Selection Buffer Mix II
¡¤ Strand Selection Enzyme Mix II
¡¤ Strand Selection Enzyme Mix III
¡¤ Adaptor Cleavage Enzyme Mix
¡¤ Amplification Buffer Mix
¡¤ Amplification Primer Mix
¡¤ Amplification Enzyme Mix
¡¤ Nuclease-free Water
¡¤ Agencourt RNAClean XP Beads
¡¤ DR Ligation Adaptor Mixes
¡¤ InDA-C Primer Mix

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