To enable the profiling of whole transcriptome at single-cell level, researchers from the Hong Kong University of Science and Technology (HKUST) developed a method to deplete rRNA in total RNA from single-cell RNA-seq libraries, termed scDASH (single-cell depletion of abundant sequences by hybridisation). Most existing scRNA-seq methods, including Smart-seq2/3 and CEL-Seq2, capture only polyadenylated mRNA to avoid the cost of sequencing abundant non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. By constructing a sgRNA library that is specific to cytoplasmic rRNA sequences and introducing it to a scRNA-seq library pool, scDASH achieved a 70% reduction in sequencing coverage of depleted rRNA genes. This in turn results in a 3.5-fold enrichment of informative reads from the rest of the transcriptome. The method increases the detection sensitivity for other RNA species, and more importantly, has opened new avenues for profiling transcripts that exist in both polyadenylated and non-polyadenylated forms such as long non-coding RNA (lncRNA).
[Figure] Total RNA from single cells is reverse-transcribed by oligo(d)T and random priming alongside. scRNA-seq libraries are amplified, pooled and subjected to scDASH treatment. Only rRNA sequences (blue) are targeted by rRNA-specific sgRNAs and cleaved by Cas9. Non-target sequences (yellow) remain intact and therefore can be enriched by PCR subsequently.