Single-Cell Semiconductor Sequencing: scRNA-Seq

It might not be an overstatement to say that virtually every neuron in our brain is unique in its position, wiring, morphological and functional properties. However, the molecular and genomic bases of this remarkable neuronal specificity are largely unknown. We simply do not know how many genes are expressed in any given neuron, how different one neuron is from another, or the scale of transcriptional changes in a neuron following physiological or pathological changes. Major limitations of existing technologies are (i) adequate quantification of all RNAs, (ii) directionality; (iii) very high cost and time-consuming sequencing needed to process numerous samples for the analysis. Here we describe a direct and efficient method (scRNA-seq) to perform unbiased capture and sequencing of RNAs in single neurons using a novel semiconductor sequencer (Ion Torrent). As a proof of concept, we selected identified motor neurons from the marine mollusk Aplysia californica as a model. Here we completed deep transcriptome analysis of two single identifiable neurons using the semiconductor and successfully validated the developed protocol using qRT-PCR and a complementary sequencing platform (Roche/454). Our protocol insures that each sequencing read corresponds to exactly one copy of the transcript within the cell. We also preserved the directionality of transcripts, providing accurate levels of both sense and anti-sense expression. Furthermore, the protocol can be universally applied to other massive parallel sequencing. We proved that each neuron has its own unique expression blueprint. Combined, our data reveal an unprecedented genomic complexity of neurons, providing the first insight into the genomic portrait of identifiable neurons directly involved in regulation of defensive behaviors.