CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians.
CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians.
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DOI：
10.1186/s12864-023-09724-4
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发表时间：
2023-10-20
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影响因子：
4.4
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单细胞rna测序（scRNA-seq）依靠PCR扩增从极少量的起始物质中检索信息。为了从大量的非聚腺苷化转录本中选择性地富集mRNA，聚(A)选择是文库制备过程中的关键步骤。然而，一些转录本，如线粒体基因，可以逃脱这种消除，使文库不堪重负。通常，这些转录本在硅中被去除，但物理损耗是否能提高单细胞中罕见转录本的检测尚不清楚。我们发现单个16S核糖体RNA在涡虫scRNA-seq数据集中广泛富集，独立于文库制备方法。为了从scRNA-seq文库中删除这个转录本，我们设计了30个单导rna，跨越它的长度。为了评估损耗的影响，我们对消除16S转录本的影响进行了并排比较，发现每个细胞检测到的基因数量大幅增加，同时几乎完全丢失了16S RNA。此外，我们系统地确定，在CRISPR处理后，文库的复杂性随着有限数量的PCR循环而增加。与硅片中16S的缺失相比，物理移除16S降低了辍学率，检索了更多的簇，并揭示了更多的差异表达基因。我们的研究结果表明，大量的转录本减少了scRNA-seq中信息转录本的检索，并扭曲了分析结果。物理去除这些污染物可以在较低的测序深度检测稀有转录本，并且在硅耗尽方面也表现出色。重要的是，这种方法可以很容易地定制，从scRNA-seq文库中删除任何丰富的转录本。在线版本包含补充材料，可在10.1186/s12864-023-09724-4获得。
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 in silico, but whether physical depletion improves detection of rare transcripts in single cells is unclear. 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 in silico depletion of 16S, physically removing it reduces dropout rates, retrieves more clusters, and reveals more differentially expressed genes. 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 in silico depletion. Importantly, this method can be easily customized to deplete any abundant transcript from scRNA-seq libraries. The online version contains supplementary material available at 10.1186/s12864-023-09724-4.