Ovation® Single Cell RNA-Seq System
The Ovation Single Cell RNA-Seq System (Part No. 0342) provides an end-to-end solution for strand-specific RNA-Seq library construction using as little as 10 picograms of total RNA, and the workflow can be completed in about 7.5 hours. The core technology used in this product enriches for non-rRNA in NGS libraries during cDNA synthesis, and can be applied to transcriptomes from a broad range of vertebrate species. The first strand cDNA synthesis is carried out using proprietary whole transcriptome primers that target non-rRNA sequences in the transcriptome. No dedicated steps are required to reduce rRNA levels. The resulting cDNA is converted to strand retained NGS libraries using reagents provided in the same kit.
The Ovation Single Cell RNA-Seq System contains reagents for production of barcoded libraries starting with total RNA. Each kit contains enough reagents for the construction of 32 RNA-Seq libraries using up to 16 unique barcoded adaptors for multiplex sequencing. These barcodes use a dedicated read (DR) barcode design.
Figure 1: Single Cell RNA-Seq Workflow
Figure 2: 5’ to 3’ Transcript Read Coverage
Graphical representation of the 5’–3’ distribution across all RefSeq genes greater than 500 bp in length of two libraries prepared with 10 pg of MAQC A (UHR) and MAQC B (brain) total RNA using the Ovation Single Cell RNA-Seq Library System. The X-axis represents the distance of the transcript from 5’ to 3’ end normalized to 100% for graphical purposes; and the Y-axis indicates the normalized coverage for each region of the transcript. Each library was sequenced on one lane of the Illumina Genome Analyzer IIx (single read sequencing, 40 bp read), generating 28.1 X 106 and 32.8 X 106 reads for UHR and brain respectively.
Figure 3: Accuracy as Detected by ERCC Spike-In Controls
The dynamic range of the Ovation Single Cell RNA-Seq System was measured using the ERCC RNA Spike-In Control Mixes (Ambion Cat # 4456740). 2.0 µL of a 1/1000 dilution of ERCC Spike-In Mix 1 was added to a 1.0 ng input of MAQC B (Brain RNA) and mapped to the ERCC reference sequences. The full set of transcripts was measured and the FPKM (Y-axis) was plotted against the actual number of transcripts converted to log2 (X-axis), which resulted in a slope of 0.986 and a Pearson correlation (R) of 0.975. The transcripts were also used to measure strand retention of >99.5%