Abstract

Takara Clontech Lunchtime Workshop - Improved cDNA Library Generation for Transcriptome Analysis from a Single Cell

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RNA-Sequencing (RNA-Seq) has revolutionized gene expression analysis even from single cells. It allows quantitative analysis of the whole transcriptome, to determine 5' and 3' ends of genes and map exon/intron boundaries. RNA-Sequencing requires reverse transcription of RNA into cDNA, the resulting cDNA can be analyzed by Next Generation Sequencing.

One powerful method for cDNA preparation is SMART™ technology (Switching Mechanism At the 5' end of the RNA Template), which utilizes the template switching activity of reverse transcriptase to enable the direct addition of a PCR adapter to the 3' end of the first-strand cDNA. The result is a single-tube protocol that enhances library amplification efficiency while minimizing the chance for contamination, making it ideal for library preparation from small amounts of starting material. Indeed, the SMARTer® Ultra™ Low RNA method allows researchers to readily obtain high quality data from a single cell or 10 pg of total RNA – the approximate amount of total RNA in a single cell.

An improved SMART protocol has been developed that is simpler and faster without compromising the quality or yield of cDNA produced. The full-length cDNA produced with this method is used as a template in library sample preparation for next generation sequencing platforms. Sequencing results for libraries created from single cells or from total RNA inputs equivalent to a single cell demonstrate that approximately 90% of the reads map to reference sequences, less than 0.5% of the total reads map to ribosomal RNA, and the average transcript coverage is uniform. Eliminating a purification step after first strand synthesis and changing the polymerase result in higher sensitivity overall with improved representation from GC-rich genes. These data indicate that the improved SMART cDNA protocol is an ideal choice for single cell transcriptome analysis.