Spatial neuron cell-type mapping in mouse brain by in situ sequencing
Recorded On: 02/07/2018
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 99 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 3 million reads per section. The read distribution for individual marker genes matches well with the Allen Brain Atlas. To turn the 99 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).
Science for Life Laboratory, Stockholm University
My research is focused on development and application of novel molecular analysis tools and systems. I have pioneered the development of methods based on DNA circularization, i.e., padlock and selector probes, as well as rolling circle amplification (RCA). The work involves fundamental studies of nuclease- and nucleic acid hybridization mechanisms, design assays based on them and integrate these into methods and systems. I have a large international network of collaborators in academia and industry, ranging from basic molecular biology and physics to clinicians and engineers. The aim is to enable new research and to bring some technologies towards diagnostic use. My group is based at Science for Life Laboratory, which is a joint cross-disciplinary research center at the Karolinska Campus, formed by Stockholm University, Karolinska Institutet, and KTH. I have authored more than 140 scientific articles that have been cited more than 7 000 times (h-index 40).