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.
Highlights
Neurons represent the basic functional units of the nervous system and are unique in many aspects, including their morphological and physiological properties (Kawaguchi, 1993; Pennartz et al, 1998; Faber et al, 2001; Hamam et al, 2002)
We describe our Patch-seq protocol, which we previously validated in a thorough analysis of human iPSC-derived neurons from healthy subjects (Bardy et al, 2016)
The approach we developed to thoroughly analyze the phenotypes of human ESC/iPSC-derived neurons in vitro using Patch-seq is innovative and sets a new standard for neurobiological analysis in the following ways:
Summary
Neurons represent the basic functional units of the nervous system and are unique in many aspects, including their morphological and physiological properties (Kawaguchi, 1993; Pennartz et al, 1998; Faber et al, 2001; Hamam et al, 2002). The minimal amount of RNA starting material in a single cell (∼10–30 pg) and the ubiquitous presence of several RNases require that an exceptional level of care must be taken to remove or inhibit any possible source of RNase contamination prior to and during lysis buffer preparation, single-neuron collection, and the pipetting of reverse-transcription mix reagents.
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