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

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

PROVIDER: PRJNA666889 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12758927_1.fastq.gz	Fastqsanger.gz
	SRR12758927_2.fastq.gz	Fastqsanger.gz
	SRR12758928.fastq.gz	Fastqsanger.gz
	SRR12758928_1.fastq.gz	Fastqsanger.gz
	SRR12758928_2.fastq.gz	Fastqsanger.gz

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

Project description: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 expending resources on profiling the types of transcripts that are usually not-of-interest, such as ribosomal RNA (rRNA). Hence, protocols that enable analysis of the whole transcriptome remain scarce. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) for rRNA depletion from single-cell total RNA-seq libraries. Our analyses show that with our single-cell DASH (scDASH), rRNAs are effectively depleted with minimal non-specificity. Importantly, the rest of the transcriptome is significantly enriched for detection as a result of liberated sequencing quota from depleted rRNA.

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Project description:The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises >90% of the total RNA molecules in a sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resources.

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

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