Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp.
Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp.
复制标题
通过膜片钳收集的 aRNA 扩增单细胞材料的稳健 RNA 测序。
DOI：
10.1038/s41598-020-58715-y
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发表时间：
2020
影响因子：
4.6
通讯作者：
Knowles,JamesA
中科院分区：
文献类型：
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作者：
Kim,JaeMunHugo;Camarena,Adrian;Walker,Christopher;Lin,MingYi;Wolseley,Victoria;Souaiaia,Tade;Thornton,Matthew;Grubbs,Brendan;Chow,RobertH;Evgrafov,OlegV;Knowles,JamesA
大多数单细胞RNA测序方案开始于从完整组织分散的单细胞。使用微流体平台能够实现分离的细胞的高通量处理。然而，组织的解离导致关于细胞位置和形态的信息的丢失，并可能改变转录组。从单细胞收集RNA的另一种方法是重新利用膜片钳记录的电生理技术。将中空贴片移液管连接到单个细胞，使得能够记录电活动，之后可以提取细胞质用于单细胞RNA-Seq（“Patch-Seq”）。由于组织未解聚，因此细胞的位置易于确定，并且细胞的形态得以保持，使得单细胞转录组与细胞位置、形态和电生理学的相关性成为可能。最近的Patch-Seq研究利用PCR扩增将核酸材料的量增加到当前测序技术所需的水平。PCR容易产生偏倚文库-特别是对于单细胞量的RNA所需的极高程度的指数扩增。我们比较了基于PCR的方法与线性扩增，并证明aRNA扩增（体外转录，IVT）是更敏感和强大的膜片钳移液器收集的单细胞RNA。
Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq (“Patch-Seq”). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries – especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitrotranscription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.