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Single Cell Sequencing for Cancer Research
T
he first study utilizing scRNA-seq examined the whole transcriptome of single cells of a four-cell stage murine embryo. A study of single cell resolution from the University of Cambridge accelerated the general understanding of gene expression and regulation under this new innovation. They were able to detect the expression of 5,720 more genes than microarray, the previous standard method for transcriptomics analysis.1 This represents a 75% increase in unique transcripts detected by the microarray technique, which clearly provides more in-depth information about the cellular states.
Tang et al. ultimately showed cell types can be clustered by tracking transcriptomic changes through scRNA-seq. While their technique was successful, there were many challenges that slowed development of the technology. The scRNA-seq libraries were generated manually in individual tubes after isolating single cells, which is a time and labor-intensive process.2 This inability to increase the
Written By Rebecca Nadler Designed By Saraswati Sridhar scale of scRNA-seq limited potential applications for several years. Researchers at Harvard Medical School innovated a method to utilize microfluidics, whereby a single cell and functional bead are contained within a droplet in an oil emulsion in order to compartmentalize cell lysing, barcoding, and reverse transcription. This procedure allowed researchers to build up large-scale sequencing libraries and is regularly used today. There is a standard methodological protocol generally used to conduct scRNA-seq.3 Arguably, the most important step of scRNA-seq is the primary isolation of viable, single cells from the cell population of interest. Establishing an unbiased, representative sample of cells is a critical step for ensuring validity of downstream data analysis. The cells are then lysed to capture RNA molecules, which are converted to complementary DNA (cDNA) via reverse transcription. In doing so, adaptor sequences are added to the ends of the cDNA molecules to serve as unique molecular barcoding tags for analysis, allowing researchers to analyze multiple samples together without ambiguity. However, reverse transcription only provides a minimal amount of cDNA, so it is amplified exponentially by polymerase chain reactions (PCR). The resulting cDNA is pooled and sequenced using Next Generation Sequencing techniques, and bioinformatic methods are utilized
Spring 2022 | PENNSCIENCE JOURNAL 27
