A well-understood technique for the targeted disruption of genes across a wide range of cell types is the use of the CRISPR/Cas9 method. In this, a single guide RNA (sgRNA) strand is used to guide the Cas9 endonuclease to create a double-strand break at a site that is complementary to the sgRNA. The resulting frameshift mutation can lead to the disruption of the open reading frame. The use of multiple sgRNAs or sgRNA libraries allows for unbiased functional genomic screens, also called CRISPR screens. More recently, pooled CRISPR screens combined with single-cell RNA sequencing (scRNA seq) have allowed the tracking of phenotypic changes in each cell to be mapped back onto the causative sgRNAs. The resulting matrix of sgRNAs versus perturbed transcriptomes provides a useful dataset to address many complex biological questions.
However, conventional scRNA seq experiments have some limitations. They are expensive due to the high costs of library preparation and next-generation sequencing. Also, they suffer from sensitivity issues due to relevant mRNAs getting outshone by more abundant ones, resulting in their sparse representation. Finally, it is difficult to monitor metrics like cell capture efficiency and multiplet rate that strongly influence the quality of sequencing.
A single-cell RNA sequencing experiment using the whole transcriptome approach (WTA) would result in the amplification of all expressed genes in an unbiased manner. However, only differentially expressed genes across cells are interesting for the study of specific phenotypes. Sequencing a complex WTA library involves spending a lot of time, effort and money on constitutively expressed mRNAs which are not informative. Therefore, it is very useful and economical to focus on a specific set of mRNAs chosen before the experiment.
In this application note, a model experiment is described that uses scRNA seq to compare an unbiased, whole transcriptome approach (WTA) with a targeted approach using a panel of mRNAs nominated a priori. The BD Rhapsody™ system is ideally suited to perform such an experiment as it allows the user to choose between running either a WTA assay or a targeted one in which selected transcripts are amplified by two nested PCR steps. The experiment shows that a targeted single-cell mRNA seq approach is cost-effective and provides a greater dynamic range of detection of transcripts.
Read the application note here.
Disclaimer: This application note is an advertisement and is not peer-reviewed.