In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia

Author:

Bakina OlgaORCID,Conrad Thomas,Mitic Nina,Vidal Ramon Oliveira,Obrusnik Tessa,Sai Somesh,Nolte Christiane,Semtner Marcus,Kettenmann HelmutORCID

Abstract

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

Funder

Helmholtz-Gemeinschaft

Berlin Institute of Health

Shenzhen Municipal Key Laboratory of Neuropsychiatric Modulation, Chinese Academy of Sciences

Immunology and Inflammation

Publisher

Public Library of Science (PLoS)

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