CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing.

Background: Single-cell RNA-sequencing (scRNA-seq) relies on PCR amplification to retrieve information from vanishingly small amounts of starting material. To selectively enrich mRNA from abundant non-polyadenylated transcripts, poly(A) selection is a key step during library preparation. However, some transcripts, such as mitochondrial genes, can escape this elimination and overwhelm libraries. Often, these transcripts are removed Results: We find that a single 16S ribosomal RNA is widely enriched in planarian scRNA-seq datasets, independent of the library preparation method. To deplete this transcript from scRNA-seq libraries, we design 30 single-guide RNAs spanning its length. To evaluate the effects of depletion, we perform a side-by-side comparison of the effects of eliminating the 16S transcript and find a substantial increase in the number of genes detected per cell, coupled with virtually complete loss of the 16S RNA. Moreover, we systematically determine that library complexity increases with a limited number of PCR cycles following CRISPR treatment. When compared to Conclusions: Our results show that abundant transcripts reduce the retrieval of informative transcripts in scRNA-seq and distort the analysis. Physical removal of these contaminants enables the detection of rare transcripts at lower sequencing depth, and also outperforms