Dataset View [GSE77564]

TitleCoupled electrophysiological recording and single-cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation
ArticleSun YE,Cheng L,Li S,Yu Y,Luo Y,He F,Yao Y,Wang J,Gao X,Zhou L,Zhang K,Chen X.Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.Protein & cell.2016 Mar
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA310792
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP069264
Overall DesginPatch-Seq of 20 hESC/hiPSC-derived neurons.
SummaryThe mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion are generated. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single-cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here, we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single-cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.
Experimental ProtocolAfter recording, the wider tip of another pipette filled with no more than 0.5 µL sterilized cell harvest solution containing (in mmol/L) 144 K-gluconate, 3 MgCl2, 0.5 EGTA and 10 HEPES (pH 7.20 and 295 mOsm/kg) was gently attached to the recorded cell, then a light suction (negative pressure) was applied, through a glass syringe (Thomas Scientific) connected to the pipette. The suction lasted until the entire cell entered the tip of the pipette. The moment the complete cell had been visually located inside the tip, the pipette was quickly removed from the bath. Content of the harvest pipette was expelled into a 0.2 mL PCR tube containing 4.5-µL pre-prepared lysis buffer.; Preparation of single-cell cDNAs was performed based on previously published protocols with modifications at several steps.
Data processingClean reads were aligned to hg19 with TopHat2 (Kim et al., 2013).; Gene expression levels were estimated by Cufflinks (Ghosh and Chan, 2016).; Genome DNA sequence and gene annotation were downloaded from iGenome (ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Homo_sapiens/UCSC/hg19/Homo_sapiens_UCSC_hg19.tar.gz).; Genome_build: GRCh37 (hg19); Supplementary_files_format_and_content: single_neuron_exp.txt: Tab-delimited text file contains gene expression level of each single neuron.
Public OnPublic on May 26 2016

Cell Groups

 hES hiPS-derived neuron[20]