Overview

The ability to sequence an entire genome by massively parallel sequencing has been applied to RNA sequencing (RNA-seq). Isolated mRNA molecules are converted to cDNA by reverse transcription and then the cDNA is prepared for sequencing with the same techniques used for genomic DNA. Sequencing the entire transcriptome at once has become a valuable tool not only for gene expression analysis but also a method to determine exon/intron boundaries, verify or amend previously annotated 5’ and 3’ gene boundaries, and identify splice variants or fusion transcripts associated with a disease. Finally, RNA-seq is a valuable tool for understanding more about the role of other types of small and large RNAs such as ncRNA, miRNA, tRNA, and ribosomal RNA.

Workflow

Once you select your particular application, a critical factor is the library preparation. Small RNA is easily degraded and available in small quantities. Selecting both the best reverse transcriptase and the right library prep is essential for obtaining high quality results. We recommend using SMARTer® Universal Low Input RNA Library Prep Kit. The ThruPLEX® technology bundled in these kits (catalogue # 634945 or 634946) is capable of amplifying very small amounts of DNA and the workflow is simple.

ThruPLEX Technology*

ThruPLEX TechnologyStep 1. ThruPLEX technology features improved repair chemistry.

Step 2. The background is reduced using double-stranded adaptors with no single-stranded tails. Blunt end ligation occurs with high-efficiency blunt-end ligation Blocked 5’ends reduce adaptor-adaptor ligation.

Step 3. Background is reduced by destroying unused adaptors after ligation.
Overall, the time to complete the process is reduced by using compatible buffers and multiplexed enzymatic reactions that do not require intermediate purifications.

Learn more about ThruPLEX DNA-seq Kit.
*Protected by US patent 7,803,550 and EP1924704

Workflow

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Clonetech’s SMARTer® Universal Low Input RNA-seq kit uses ThruPLEX technology to amplify even low amounts of input DNA.