Wednesday, October 13, 2010

Obtaining optimal RNA-Seq library quality

The new ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina-compatible) prepares directional, ligation-free RNA-Seq libraries in less than 3 hours, from rRNA-depleted or poly(A)+ RNA. We compared the quality of ScriptSeq libraries prepared using different methods to treat the total RNA.

We used Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), and total RNA isolated from FFPE breast cancer tissue as starting material. The specified samples were treated with either the Ribo-Zero™ Kit, a competitive rRNA-removal kit (Company A), or a commercial oligo(dT)-based mRNA enrichment kit. For UHRR and BrRR, ScriptSeq libraries were prepared from 50-ng aliquots of the resulting rRNA-depleted or poly(A)-enriched RNA. For FFPE samples, the entire amount of rRNA-depleted RNA recovered from 500 ng total RNA input was used to prepare the libraries. The di-tagged cDNA reactions were amplified by PCR for either 10 cycles (UHRR and BrRR) or 12 cycles (FFPE) followed by Exo I digestion. Each RNASeq library was purified using MinElute (Qiagen) and recovered in 15 μl of Elution Buffer. Replicate reactions were pooled and examined using a Bioanalyzer (Agilent). Single-lane, 54-nt unidirectional sequencing reads were obtained for each library using an Illumina GAII sequencer, and sequence analysis was performed using Bowtie.

Summary of sequencing data from ScriptSeq™ libraries (click to enlarge figure). Libraries were prepared as described above from Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), or RNA extracted from FFPE breast cancer tissue (FFPE). The indicated method of rRNA removal or mRNA enrichment was used.

As seen in the figure, optimal RNA-Seq results were obtained with the Ribo-Zero Kit for rRNA removal, even from substantially degraded RNA (FFPE sample).

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