Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH

Danson S C Loi; Lei Yu; Angela R Wu

doi:10.7717/peerj.10717

Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH

PeerJ. 2021 Jan 15:9:e10717. doi: 10.7717/peerj.10717. eCollection 2021.

Authors

Danson S C Loi¹, Lei Yu¹, Angela R Wu^{1

2

3}

Affiliations

¹ Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
² Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
³ Hong Kong Branch of Guangdong Southern Marine Science and Engineering Laboratory (Guangzhou), Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

Abstract

A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.

Keywords: CRISPR; Single-cell transcriptomics; rRNA depletion; scRNA-seq.

Grants and funding

This work was funded by HKUST’s start-up and initiation grants (Hong Kong University Grants Committee), the Hong Kong Research Grants Council Early Career Support Scheme (RGC ECS 26101016), the Hong Kong Epigenomics Project (LKCCFL18SC01-E), HKUST BDBI Labs, the Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (SMSEGL20SC01), as well as by a donation from the Chou Hoi Shuen Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.