CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing.
CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing.
复制标题
基于 CRISPR/Cas9 的 16S 核糖体 RNA 消耗提高了单细胞 RNA 测序的文库复杂性。
DOI：
10.1101/2023.05.25.542286
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Adler,CarolynE
中科院分区：
文献类型：
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作者：
Wang,Kuang-Tse;Adler,CarolynE
背景单细胞RNA测序（scRNA-seq）依赖于PCR扩增来从消失的少量起始材料中检索信息。为了从丰富的非聚腺苷酸化转录物中选择性地富集mRNA，聚腺苷酸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，可以逃避这种消除和压倒文库。通常情况下，这些成绩单被删除在硅片，但无论是物理消耗提高检测罕见的成绩单在单cells.ResultsWe发现，一个单一的16 S核糖体RNA是广泛富集在涡虫scRNA-seq数据集，独立的库制备方法。为了从scRNA-seq文库中消除这种转录物，我们设计了30个跨越其长度的单向导RNA。为了评估消除的影响，我们对消除16 S转录物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，加上16 S RNA几乎完全丢失。此外，我们系统地确定了CRISPR处理后文库复杂性随着有限数量的PCR循环而增加。当在硅片耗尽16 S相比，物理删除它降低了辍学率，检索更多的集群，并揭示了更多的差异表达的genes.ConclusionsOur研究结果表明，丰富的成绩单减少检索scRNA-seq中的信息转录本和扭曲的分析。这些污染物的物理去除使得能够在较低的测序深度下检测稀有转录物，并且还优于计算机模拟消除。重要的是，这种方法可以很容易地定制，以消除scRNA-seq文库中任何丰富的转录本。
BackgroundSingle-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.ResultsWe 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.ConclusionsOur 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.