Spatial neuron cell-type mapping in mouse brain by in situ sequencing
Xiaoyan Qian1, Nathan Skene2, Ana Muñoz Manchado2, Jens Hjerling-Leffler2, Kenneth D. Harris3, Mats Nilsson1
1Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University; 2Department of Medical Biochemistry and Biophysics, Karolinska Institutet; 3Institute of Neurology, University College London
Single-cell RNA-seq (scRNA-seq) is a powerful tool to classify cells to molecularly defined cell types. However, the information about absolute frequency of cells and exact spatial location the within the original tissue is lost. The brain is the most complex tissue in mammals with respect to the number of different cell-types and the way they are arranged locally and through long range cell-to-cell connections. Here, we demonstrate that in situ sequencing (Ke et al., Nat.Meth., 2013) can be used to build a cell-type spatial map of 100 000s of cells in sections of mouse brain. We use in situ sequencing to map the activity of 84 marker genes within single cells across sections of mouse brains. The marker genes are selected to identify neurons in cortex and hippocampus as defined by scRNAseq. In a single experiment on a single standard microscopy slide, we can analyse four coronal brain sections from adult mice. Each section contains around 100,000 cells and we generate about 1.5 million reads per section. The read distribution for individual marker genes matches well with the Allen Brain Atlas. To turn the 84 molecular distributions into cell-types, we use a probabilistic approach to assign identity to individual cells based on comparison with the profiles of 35 cell types as defined by scRNA-seq. The sensitivity of the approach is demonstrated by our identification of rare Pvalb-expressing cells among pyramidal cells in stratum pyramidale, and Cck-positive cells, in stratum radiatum.
Ke, R., Mignardi, M., Pacureanu, A., Svedlund, J., Botling, J., Wahlby, C. & Nilsson, M. In situ sequencing for RNA analysis in preserved tissue and cells Nat. Methods 10, 857-860 (2013).