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Front Mol Neurosci. 2018 Aug 10;11:261. doi: 10.3389/fnmol.2018.00261. eCollection 2018.

Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells.

Author information

1
Laboratory for Human Neurophysiology and Genetics, South Australian Health and Medical Research Institute (SAHMRI) Mind and Brain, Adelaide, SA, Australia.
2
The Lieber Institute for Brain Development, Baltimore, MD, United States.
3
Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, MD, United States.
4
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, United States.
5
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
6
Laboratory of Genetics, Salk Institute for Biological Studies, Sanford Consortium for Regenerative Medicine, La Jolla, CA, United States.
7
Flinders University College of Medicine and Public Health, Adelaide, SA, Australia.

Abstract

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently, advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method, referred to as Patch-seq, enables a thorough, multimodal profiling of neurons and permits us to expose the links between functional properties, morphology, and gene expression. Here, we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects, but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders.

KEYWORDS:

cellular phenotyping; electrophysiology; human neuron transcriptome; induced pluripotent stem cell (iPSC); neuronal diversity; patch clamping; patch-seq; single-cell RNA-seq

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