A critical assessment of single-cell transcriptomes sampled following patch-clamp electrophysiology

Patch-seq, combining patch-clamp electrophysiology with single-cell RNA-sequencing (scRNAseq), enables unprecedented single-cell access to a neuron’s transcriptomic, electrophysiological, and morphological features. Here, we present a systematic review and re-analysis of scRNAseq profiles from 4 recent patch-seq datasets, benchmarking these against analogous profiles from cellular-dissociation based scRNAseq. We found an increased likelihood for off-target cell-type mRNA contamination in patch-seq, likely due to the passage of the patch-pipette through the processes of adjacent cells. We also observed that patch-seq samples varied considerably in the amount of mRNA that could be extracted from each cell, strongly biasing the numbers of detectable genes. We present a straightforward marker gene-based approach for controlling for these artifacts and show that our method improves the correspondence between gene expression and electrophysiological features. Our analysis suggests that these technical confounds likely limit the interpretability of patch-seq based single-cell transcriptomes. However, we provide concrete recommendations for quality control steps that can be performed prior to costly RNA-sequencing to optimize the yield of high quality samples.