Zhilka Neurogenetic institute, University of Southern California, 1501 San Pablo St, Los Angeles, CA, 90033, USA.
University of California, San Diego 9500 Gilman Dr, La Jolla, CA, 92093, USA.
Sci Rep. 2020 Feb 6;10(1):1979. doi: 10.1038/s41598-020-58715-y.
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 vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.
大多数单细胞 RNA 测序方案都是从完整组织中分散的单细胞开始的。使用微流控平台可以实现对分离细胞的高通量处理。然而，组织的解离会导致细胞位置和形态信息的丢失，并可能改变转录组。另一种从单细胞中收集 RNA 的方法是重新利用膜片钳记录的电生理技术。将中空的膜片钳管附着到单个细胞上，在记录电活动后，可以提取细胞质进行单细胞 RNA-Seq（“Patch-Seq”）。由于组织没有被分散，因此很容易确定细胞的位置，并且可以保持细胞的形态，从而可以将单细胞转录组与细胞位置、形态和电生理学相关联。最近的 Patch-Seq 研究利用 PCR 扩增将核酸物质的量增加到当前测序技术所需的水平。PCR 容易产生偏向性文库 - 特别是对于单细胞 RNA 所需的极其高的指数扩增程度。我们比较了基于 PCR 的方法与线性扩增，并证明了通过膜片钳管收集的单细胞 RNA 的 aRNA 扩增（体外转录，IVT）更灵敏和稳健。
