Interrogate the Whole Transcriptome and Small RNA on the SOLiD® Sequencing System

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Figure 1. Click to enlarge. The SOLiD® Total RNA-Seq Kit protocol. Whole transcriptome libraries and small RNA libraries can be constructed using the SOLiD® Total RNA-Seq Kit and the respective protocol. To prepare RNA for a whole transcriptome library, start with 500 ng total RNA, 200–500 ng total RNA depleted of ribosomal RNA (RiboMinus™ Eukaryote Kit for RNA-Seq, Cat. No. A1083708, or RiboMinus™ Plant Kit for RNA-Seq, Cat. No. A1083808) or 100–500 ng PolyA RNA, and fragment. To prepare a small RNA library, small RNA should be purified and enriched from good-quality total RNA followed by determination on input quantity. Both library protocols follow the same workflow for construction with the ligation of SOLiD® adaptors, reverse transcription, and size selection for subsequent amplification. After assessing the amplified DNA for yield and size distribution, the library is ready for ePCR on the SOLiD® System.

The SOLiD® Total RNA-Seq Kit is designed as one complete solution, providing streamlined protocols (Figure 1) for whole genome discovery or small noncoding RNA analysis. The kit optimally combines all necessary reagents to enable hypothesis-neutral discovery of coding RNA, noncoding RNA, novel transcripts, alternate splicing, small RNAs, and isoforms, all while maintaining accurate sequence representation and preserving strand direction.

 

One Complete Solution for RNA Sequencing on the SOLiD® System

Specifically designed for use with the SOLiD® System, the SOLiD® Total RNA-Seq Kit contains enough reagents to process up to 12 samples. Either whole transcriptome libraries or small RNA libraries can be barcoded using SOLiD® RNA Barcoding Kits, allowing the respective pooling of size-selected libraries for more efficient and cost-effective analysis using a single slide.

  • Convenient—streamlined workflow for either whole transcriptome or small RNA analysis
  • Revealing—designed for strand-specific expression analysis where other kits fail to differentiate
  • Qualified—compatible with the SOLiD® 4 System to perform paired-end sequencing
  • Versatile—allows multiple types of RNA input
  • Flexible—enables you to increase sample numbers cost effectively with SOLiD® RNA Barcoding Kits

SOLiD® Total RNA-Seq Kit Maintains Strand Specificity

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Figure 2. (Click to enlarge.) Ligase Enhanced Genome Detection (LEGenD™) technology.

Using Life Technology’s proprietary Ligase Enhanced Genome Detection (LEGenD™) technology (Figure 2), RNA is converted into a library of double-stranded cDNA molecules. Genomic strand specificity is preserved through the simultaneous addition of adaptors in a directional manner.

High-throughput sequencing has revealed that transcripts are also synthesized from the antisense strand of DNA. In addition, other types of noncoding RNA have been shown to represent a significant fraction of the genome and to be transcribed from both strands of DNA. For this reason, it is important to know to which strand of DNA each RNA transcript maps (Figure 3). To learn more about the importance of strand specificity, click here.

Figure 3. Read coverage of converging transcripts PRKCSH and ELAVL3 demonstrates strand determination using the SOLiD® Total RNA-Seq Kit.
SOLiD® sequencing tags generated from HBR RNA map to specific strands in a region of the human genome where the 3′ ends of two genes, PRKCSH and ELAVL3, converge. Sense and antisense expression is indicated in green and red colors, respectively. The distinct pattern of green and red regions demonstrates that PRKCSH

Whole Transcriptome Analysis

The whole transcriptome analysis protocol enables you to rapidly construct strand-specific libraries from total RNA, ribosomal-depleted total RNA, or Poly(A+) RNA. Because you are not limited to using Poly (A+) RNA, you can perform a more thorough investigation of trancriptome complexity. To remove ribosomal RNA, Invitrogen’s RiboMinus™ RNA-Seq is recommended (available for both eukaryotic and plant rRNA removal).

  • Hypothesis-free global expression analysis—detect known and unknown RNA molecules including exons, fusion transcripts, mutations, and SNP detection
  • Maintenance of genomic DNA strand specificity
  • Start with as little as 100 ng of Poly(A+) RNA, 200 ng of rRNA depleted total RNA, or 500 ng total RNA
  • Simple, robust 2-day protocol for library construction

Small RNA Analysis

Small noncoding RNAs play a key role in regulating a variety of biological processes, including developmental timing, cellular differentiation, and tumor progression. Small RNAs are typically 18–40 nucleotides in length and belong to one of a variety of small RNA classes such as microRNA (miRNA), short interfering RNA (siRNA), and piwi-interacting RNA (piRNA).

  • Hypothesis-free global expression analysis—capable of detecting all small RNA in a single assay
  • Maintenance of genomic DNA strand specificity
  • Highly sensitive—interrogate low expression levels from a limited quantity of total RNA (10-fold less than other commercially available kits)
  • Simple, single-day protocol for library construction
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