Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp

Jae Mun Hugo Kim, Adrian Camarena, Christopher Walker, Ming Yi Lin, Victoria Wolseley, Tade Souaiaia, Matthew Thornton, Brendan Grubbs, Robert H. Chow, Oleg V. Evgrafov, James A. Knowles

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number1979
JournalScientific reports
Volume10
Issue number1
DOIs
StatePublished - Dec 1 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

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