Journal: Frontiers in Molecular Neuroscience
Article Title: Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells
Figure Lengend Snippet: Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the cDNA is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
Article Snippet: The QC pipeline described here is based on (i) expression profiling of common housekeeping genes and differentially abundant exogenous reference transcripts (ERCC spike-in controls), (ii) fluorometric quantitation of cDNA yield (Qubit), and (iii) qualitative analysis of cDNA fragment profiles (Agilent Bioanalyzer).
Techniques: Functional Assay, Patch Clamp, Imaging, Transferring, Lysis, Polymerase Chain Reaction, Amplification, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, RNA Sequencing Assay