Strand Orientation: Figure 1

Ambion®, the leader in RNA analysis technologies, offers the first kit to streamline high-throughput sequencing of the whole transcriptome and small RNAs, while maintaining strand specific, on the Illumina® GAII platform. The Ambion® RNA-Seq Library Construction Kit compatible with the Illumina® GAII platform provides, in an optimized kit, all of the necessary reagents to discover coding RNAs, non-coding RNAs, and small RNAs and isoforms, as well as to show alternative splicing events. Based on the robust technology used in the SOLiD® Total RNA-Seq Kit, the Ambion® RNA-Seq Library Construction Kit compatible with the Illumina® GAII platform allows greater flexibility in RNA starting samples and improves sequencing results from the Illumina® GAII platform when compared to data from sequencing runs using the instrument manufacturer’s kits.

  • Convenient—streamlined workflow interrogates both the whole transcriptome and small RNAs using one kit
  • Versatile—use with total RNA, rRNA-depleted total RNA, mRNA, and small RNA
  • Revealing—preserves strand specific relative to the chromosomal strand, aligning all mapped reads in the direction of transcription

Whole transcriptome and small RNA in one kit

With flexible input requirements and the ability to sequence a whole transcriptome or small RNA, the Ambion® RNA-Seq Library Construction Kit compatible with the Illumina® GAII platform is a convenient solution that appeals to the needs of any RNA-Seq experiment on the Illumina® GA II platform (Figure 1).

Enlarge Image
Figure 1. Click to enlarge.

Figure 1. The Ambion® RNA-Seq Library Construction Kit protocol. Whole transcriptome libraries and small RNA libraries can be constructed using the Ambion® RNA-Seq Library Construction Kit and the respective protocol. To prepare RNA for a whole transcriptome library, start with total RNA, total RNA depleted of ribosomal RNA (use RiboMinus™ Eukaryote Kit for RNA-Seq, Cat. No. A1083708, or RiboMinus™ Plant Kit for RNA-Seq, Cat. No. A1083808), or poly(A) RNA, and fragment it. To prepare a small RNA library, small RNA should be purified and enriched from high-quality total RNA, followed by determination of input quantity. Both library protocols follow the same workflow for construction with the ligation of adaptors, reverse transcription, and size selection for subsequent amplification. After assessment of the amplified DNA for yield and size distribution, the library is ready for cluster generation on the Illumina® GAII platform

To perform whole transcriptome analysis, construct an RNA library compatible with paired-end sequencing in just 2 days, starting with as little as 100 ng of poly(A) RNA, 200 ng of rRNA-depleted RNA, or 200 ng of total RNA. Since each library is constructed to preserve information on chromosomal strand location, subsequent sequencing can detect known and unknown transcripts, including alternative splicing products, fusion transcripts, and SNPs (Figure 2).

Figure 2. Click to enlarge.

Figure 2. Whole transcriptome mapping performance. RNA-Seq libraries were generated using the MAQC human brain reference (HBR) and universal human reference (UHR) poly(A) RNA and sequenced on the Illumina® GAII instrument. Resulting sequence reads were mapped to RefSeq annotated transcripts (version 40), then tallied per sample. Dark green bars indicate the total number of reads detected, and light green bars indicate the number of reads that map to RefSeq transcripts, which in most cases represent approximately 50% of the total detected reads.

A small RNA protocol enables the rapid construction of small RNA (microRNA ~10-40nt) libraries starting with total RNA or enriched small RNA. The Ambion® RNA-Seq Library Construction Kit compatible with the Illumina® GAII platform typically yields at least 50% mapping of all reads to miRBase (Figure 3).

Figure 3. Click to enlarge.

Figure 3. MicroRNA mapping performance. Lung and placental RNA were used to make small RNA libraries by capturing microRNAs (miRNAs) in order to examine relative miRNA detection between samples. Each sample was sequenced in duplicate on the Illumina® GAII instrument and subsequently mapped to a miRBase 13.0 reference containing known mature miRNA sequences. Dark blue bars indicate the total number of reads detected, and light blue bars indicate the number of reads that map to miRBase. Typically, placental RNA is more enriched for miRNAs than lung RNA, with at least 50% of all reads for both sample types successfully mapping to miRBase miRNAs.

Preserving Strand Specific Reveals New Data

Standard methods for constructing an RNA-Seq library fail to distinguish which strand a transcript is transcribed from. Consequently, sequencing data show equal levels of transcription from both strands. Without differentiation of areas of transcription between the two strands, valuable information is masked, such as the elucidation of antisense transcripts with regulatory function (which are different from those of the sense strand) and areas where alternative splicing or fusion transcript events occur between the two strands. In the example shown below (Figure 4), two distinct, adjacent, divergent transcripts are detected with strand-specific, genome-aligned reads. Without this strand information, the data would have appeared to represent a single transcriptional unit.

Enlarge Image
Figure 4. Click to enlarge.

Figure 4. Transcript coverage profiles generated from mapped reads derived from strand-specific and non–strand-specific library construction methods. Sequencing libraries were generated using a library construction method that maintains strand specific(top panel) and another method that does not maintain strand specific(bottom panel), using the MAQC human brain reference poly(A) RNA. Resulting sequence reads were mapped and then formatted for display in the UCSC Genome Browser. PRKCSH and ELAVL3 are transcribed on the forward (<) and reverse (>) chromosomal strands, respectively. The method using the Ambion® kit, which maintains strand specific, clearly shows strand-specific alignment and makes it easy to distinguish between the two genes. On the other hand, the method that does not maintain strand specificity makes the process of distinguishing between the two genes more difficult, as reads align with equivalent counts on both strands of all reads for both sample types that successfully map to miRBase miRNAs. 

Based on the same proprietary technology used in the SOLiD® Total RNA-Seq Kit, the Ambion® RNA-Seq Library Construction Kit compatible with the Illumina® GAII platform most effectively constructs an RNA-Seq library that differentiates strands. Unlike other methods that ligate adaptors to double-stranded cDNA, the technology used in the Ambion® kit simultaneously attaches the 3’ and 5’ adaptors in a directional manner, reducing ligation and clean-up steps (Figure 5).

Enlarge Image
Figure 5. Click to enlarge.

Figure 5. Ambion® Ligase Enhanced Genome Detection (LEGenD™) technology. Using Life Technologies’ proprietary Ambion® Ligase Enhanced Genome Detection (LEGenD™) technology, 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.

Illumina is a registered trademark of Illumina, Inc. Illumina, Inc. is not affiliated, sponsored, or endorsed by Life Technologies Corporation.

Related Products

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Support

NEW   Next-Generation Sequencing Support Center
Find tips, troubleshooting help, and resources for your next-gen sequencing applications.