GSE112177
A guide to single-cell transcriptomics in adult rodent brain: the medium spiny neuron transcriptome revisited
Recent advances in single-cell technologies are paving the way to a comprehensive understanding of the cellular complexity in the brain. Protocols for single-cell transcriptomics combine a variety of sophisticated methods in order to isolate heavily interconnected and heterogeneous neuronal cell types in a relatively intact and healthy state. The emphasis of single-cell transcriptome studies has thus far been on comparing library generation and sequencing techniques that enable measurement of the minute amounts of starting material from a single cell. However, in order for data to be comparable, standardized cell isolation techniques are essential. Here, we analyzed and simplified methods for the different steps critically involved in single-cell isolation from brain. These include highly region-specific cellular labelling compatible with use of stereotaxic coordinates, enzymatic digestion, tissue trituration, and improved methods for efficient fluorescence-activated cell sorting in samples containing high degree of debris from the neuropil. The methods are exemplified using medium spiny neurons (MSN) from dorsomedial striatum, a cell type that is clinically relevant for disorders of the basal ganglia, including psychiatric and neurodegenerative diseases. We present single-cell RNA sequencing (scRNA-Seq) data from D1 and D2 dopamine receptor expressing MSN subtypes. We illustrate the need for single-cell resolution by comparing to available population-based gene expression data of striatal medium spiny neuron subtypes. Our findings contribute towards standardizing important steps of single-cell isolation from adult brain tissue to increase comparability of data. Furthermore, our data newly define the transcriptome of medium spiny neurons at unprecedented resolution and identify novel subtype specific marker genes in this important cell type Overall design: 40 Drd1 and 40 Drd2 expressing neurons from double-transgenic animals (Drd1a-TdTomato X Drd2-EGFP) were sequenced by scRNA-Seq. Cells were FACS sorted into single wells 96-well plates and selected based on their fluorescence intensity (TdTomato > Drd1 expressing, EGFP > Drd2 expressing). Single-cell Samples were processed with SMARTer Ultra Low Input RNA Kit for Sequencing – v3 (Clontech Laboratories) and sequenced on a MiSeq instrument (Illumina)