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Encore® Complete RNA-Seq Library Systems
The Encore® Complete RNA-Seq Library Systems provide an end-to-end solution for strand-specific RNA-Seq library construction using as little as 100 ng of total RNA. The core technology used in this product enriches for non-rRNA in NGS libraries during cDNA synthesis, and can be applied to transcriptomes extracted from a broad range of higher eukaryotes. The first strand cDNA synthesis is carried out using proprietary primers to create double-stranded cDNA which retains RNA strand information. No dedicated steps are required to reduce rRNA levels. The resulting cDNA is converted to NGS libraries using reagents and adaptors provided in the same kit. The Encore Complete RNA-Seq Multiplex Systems provide optional barcoding to further optimize efficiencies and cost savings in transcriptome sequencing.
The Encore Complete RNA-Seq Library Systems have been designed for strand-specific expression analysis by incorporation of a nucleotide analogue during the second strand cDNA synthesis, and subsequent ligation, to a pair of double-stranded 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:
The Encore Complete RNA-Seq Library System (Part No. 0311) contains reagents for production of non-barcoded libraries starting with total RNA, while the Encore Complete RNA-Seq IL Multiplex System 1-8 (Part No. 0312) and Encore Complete RNA-Seq IL Multiplex System 9-16 (Part No. 0313) each provide eight unique barcoded adaptors for multiplex sequencing. In combination these latter two kits enable up to 16-plex sequencing. The Encore Complete RNA-Seq DR Multiplex System 1-8 (Part No. 0333) and Encore Complete RNA-Seq DR Multiplex System 9-16 (Part No. 0334) offer the same level of multiplexing but use a dedicated read (DR) barcode design.
Figure 2:
Strand-Specific Read Coverage: Total RNA from MAQC B (Human brain) was amplified using either the Ovation® RNA-Seq System V2 (Part No. 7102) or poly(A)+ RNA selected, or processed using the Encore Complete RNA-Seq Library System. In the cases of Ovation RNA-Seq System V2 and poly(A)+ samples, the resulting double-stranded cDNA was input to the Encore NGS Multiplex System I (Part No. 301) to construct NGS libraries. Single-read sequencing results were obtained using the Illumina Genome Analyzer IIx platform. Sequencing read coverage plots are shown for PTMS and MLF2 transcripts. The results show read coverage across all exons, and illustrate how the sequencing reads segregate to the plus (+) strand for PTMS and the minus (-) strand for MLF2 when using the strand retention capability of the Encore Complete RNA-Seq Library System.