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A rapid, directional RNA-seq library preparation workflow for Illumina® sequencing

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Nature Methods
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Most current RNA-seq library preparation methods are time-consuming, multistep processes. We describe a workflow that includes the Ribo-Zero and ScriptSeq v2 Kits that enables researchers to go from total RNA to cluster-ready RNA-seq libraries in less than 1 d. The RNA-seq libraries produced are virtually free of contaminating ribosomal RNA (rRNA) and provide for directional paired-end and multiplex sequencing on Illumina® sequencing platforms.

At a glance


  1. Overview of the ScriptSeq v2 RNA-seq library preparation method.
    Figure 1: Overview of the ScriptSeq v2 RNA-seq library preparation method.
  2. Consistent coverage of ScriptSeq v2 RNA-seq libraries from varying amounts of input RNA.
    Figure 2: Consistent coverage of ScriptSeq v2 RNA-seq libraries from varying amounts of input RNA.


Massively parallel sequencing of cDNA produced from RNA (RNA-seq) has become a widely accepted alternative to microarrays for transcript profiling and analysis of new transcripts, new isoforms, alternative splice sites, rare transcripts and cSNPs. Current RNA-seq library preparation methods comprise preparing rRNA-depleted or poly(A)-enriched RNA followed by RNA fragmentation, cDNA synthesis, adaptor ligation and multiple cleanup steps. These methods are generally time consuming, requiring about 1.5 d and significant hands-on time. They typically produce nondirectional libraries.

We describe a greatly improved RNA-seq library preparation workflow that overcomes challenges associated with conventional methods. The workflow includes highly efficient rRNA removal (Ribo-Zero technology) followed by a rapid, ligation-free cDNA synthesis procedure for preparing directional RNA-seq libraries (ScriptSeq v2 technology).

rRNA removal

Ribo-Zero kits use a hybridization-capture process that removes >99% of cytoplasmic rRNA (and optionally, mitochondrial rRNA) from 1 μg to 5 μg of intact, partially degraded or formalin-fixed paraffin-embedded (FFPE) total RNA samples (Benes, V. et al. Ribo-Zero Gold Kit: improved RNA-seq results after removal of cytoplasmic and mitochondrial ribosomal RNA. Nat. Methods Application Note, 8, iii-iv, November 2011). The single-pass procedure can be completed in 1.5 h.

Rapid RNA-seq library preparation

The ScriptSeq v2 RNA-Seq Library Preparation Kit uses a patented terminal-tagging process (Fig. 1) to generate directional RNA-seq libraries in approximately 4 h. Briefly, 500 pg to 50 ng of Ribo-Zero–treated or poly(A)+ RNA is fragmented and reverse transcribed using random primers containing a 5′-tagging sequence. The 5′-tagged cDNA is then tagged at its 3′ end by the terminal-tagging reaction to yield di-tagged, single-stranded cDNA. Following purification, the di-tagged cDNA is amplified by limited-cycle PCR, which completes the addition of the Illumina adaptor sequences, amplifies the library for subsequent cluster generation and adds an optional Illumina Index or user-defined barcode. The amplified RNA-seq library is purified and is ready for cluster generation and sequencing.

Figure 1: Overview of the ScriptSeq v2 RNA-seq library preparation method.

Directionality and coverage of ScriptSeq v2 libraries

We prepared ScriptSeq v2 libraries using total RNA from various sources after treatment with the Ribo-Zero Kit (Human/Mouse/Rat) or after poly(A) enrichment. In addition, we compared intact and fragmented RNA samples. All libraries were sequenced on an Illumina GAIIx sequencer. As shown in Table 1, the number of reads passing filter and Q30 scores1, 2 for ScriptSeq v2 libraries are within normal limits for Illumina sequencing.

Table 1: Summary of RNA-seq metrics from ScriptSeq v2 libraries

The random-primed cDNA synthesis and terminal-tagging steps employed by the ScriptSeq v2 procedure add unique sequence tags to the 5′ and 3′ ends of the di-tagged cDNA that is synthesized. These unique tags permit >98% directional sequencing reads (Table 1). Using the Illumina sequencing primers, a single-read ScriptSeq v2 library generates the sequence corresponding to the sense strand of the original RNA molecule and a paired-end (reverse-end) read generates the antisense sequence of the original RNA molecule.

Figure 2 shows the sequence coverage of 600 transcripts in a ScriptSeq v2 RNA-seq library produced from poly(A)+ universal human reference (UHR)RNA and sequenced using single-end reads on an Illumina GAIIx. The data demonstrate consistent sequence coverage using 500 pg to 50 ng of input RNA.

Figure 2: Consistent coverage of ScriptSeq v2 RNA-seq libraries from varying amounts of input RNA.


The RNA-seq library preparation workflow presented here enables researchers to go from intact or fragmented total RNA samples to cluster-ready RNA-seq libraries in less than 1 d. Ribo-Zero technology provides highly efficient removal of rRNA from both intact and fragmented RNA samples, including FFPE RNA. ScriptSeq v2 library preparation technology generates ligation-free, directional RNA-seq libraries for single-read, paired-end read and multiplexed Illumina sequencing. The RNA-seq libraries produced exhibit high quality, strong directionality and good transcript coverage.


  1. Ewing, B. et al. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175185 (1998).
  2. Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II Error probabilities. Genome Res. 8, 186194 (1998).

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We are grateful to N.J. Maness (University of Wisconsin AIDS Vaccine Laboratory) for rhesus macaque library samples, J. Hitchen (Epicentre) for technical assistance and C. Kinross (Epicentre) for data analysis.

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  1. Epicentre (an Illumina company), Madison, Wisconsin, USA.

    • Jim Pease &
    • Roy Sooknanan

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This article was submitted to Nature Methods by a commercial organization and has not been peer reviewed. Nature Methods takes no responsibility for the accuracy or otherwise of the information provided.

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