Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization

Abstract

The Patch-seq approach is a powerful variation of the standard patch clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at a scale and quality that can be integrated with high-throughput dissociated cell transcriptomic data, we have optimized the technique by identifying and refining key factors that contribute to the efficient collection of high-quality data. To rapidly generate high-quality electrophysiology data, we developed patch clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized a substantial improvement in transcriptomic data quality when the nucleus was extracted following the recording. For morphology success, the importance of maximizing the neurons membrane integrity during the extraction of the nucleus was much more critical to success than varying the duration of the electrophysiology recording. We compiled the lab protocol with the analysis and acquisition software at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data compatible with recent large scale publicly available Allen Institute Patch-seq datasets.

Local Connectivity and Synaptic Dynamics in Mouse and Human Neocortex

“…We confirmed that, in at least one case, both cells in the pair had axons extending into L1 and sparsely spiny dendrites, consistent with Sst neurons (Fig S3B). We performed follow-up experiments utilizing the Patch-seq method (41) and further confirmed that cells which transcriptionally mapped to the Sst subclass do form connections with each other (3 connections found out of 47 probed, Fig S3C).…”

“…With the recent advancements in the segmentation and annotation of large volumes from electron-microscopy (Turner et al, 2020), the opportunity to reconcile connectivity measurements from functional studies like ours with anatomical measurements is on the horizon. The combination of patchSeq (Gouwens et al, 2020;Lee et al, 2020) and multipatch experiments creates the opportunity to gain insights into local connectivity among transcriptomic cell types. A straightforward path is the continuation of the pipeline to explore other brain regions' microcircuitry to reveal the extent to which circuits are either conserved or specialized.…”

“…Methods for nuclei extraction and processing are similar to previous patchSeq studies (Gouwens et al, 2020;Lee et al, 2020). At the end of the experiment, pipettes were adjusted to the soma center or placed near the nucleus, if visible.…”

“…Materials used to make and store aCSF V were cleaned thoroughly before use. In patchSeq experiments, a subset of stimuli were collected to limit progressive cell swelling associated with the addition of RNAse to the internal solution (41) .…”

In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives

“…To link the molecular and physiological properties of single cells, researchers applied a combined method of whole-cell recording and single-cell RNA sequencing (scRNA-seq) to individual neurons in acute slices in early studies and named this method “patch-seq” [ 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 ]. The basic protocol of patch-seq is as follows: researchers perform whole-cell recording from individual neurons from living tissue slices or even intact animals and aspirate the cell contents through a patch pipette for subsequent RNA sequencing.…”