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

Area of Science:

• Neuroscience
• Molecular Biology
• Genomics

Background:

• Standard single-cell RNA sequencing (scRNA-Seq) protocols require tissue dissociation, leading to loss of spatial and morphological information.
• This dissociation process can also introduce transcriptomic alterations, limiting the comprehensive analysis of cellular function.
• Patch clamp recording, an electrophysiology technique, offers a way to study individual cells within intact tissue.

Purpose of the Study:

• To evaluate an alternative single-cell RNA collection method by repurposing patch clamp recording for RNA sequencing (Patch-Seq).
• To compare the efficacy of different RNA amplification methods for Patch-Seq, specifically comparing PCR-based amplification with linear amplification (aRNA IVT).
• To determine the optimal amplification strategy for sensitive and robust transcriptomic analysis from single cells isolated via patch clamp.

Main Methods:

• Single cells were isolated using patch clamp recording from intact tissue, allowing for simultaneous electrophysiological measurements.
• Cytoplasmic contents, including RNA, were extracted from individual patched cells.
• RNA was amplified using either traditional PCR-based methods or linear amplification via in vitro transcription (aRNA IVT).
• The amplified RNA was then subjected to single-cell RNA sequencing.

Main Results:

• Patch-Seq successfully preserved cell location and morphology, enabling correlation with transcriptomic and electrophysiological data.
• PCR-based amplification exhibited biases, particularly with the high amplification factors needed for single-cell RNA.
• aRNA amplification (IVT) demonstrated superior sensitivity and robustness compared to PCR for analyzing single-cell RNA obtained through Patch-Seq.
• Linear amplification provided a more accurate and reliable representation of the original transcriptome.

Conclusions:

• Patch-Seq is a valuable method for integrating transcriptomic, morphological, and electrophysiological data from single cells within their native tissue environment.
• Linear RNA amplification (aRNA IVT) is the preferred method for amplifying RNA from Patch-Seq experiments due to its enhanced sensitivity and reduced bias compared to PCR.
• This optimized Patch-Seq approach provides a more comprehensive understanding of cellular heterogeneity and function in complex tissues.