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

Bakina, O.; Conrad, T.; Mitic, N.; Vidal, R.O.; Obrusnik, T.; Sai, S.; Nolte, C.; Semtner, M.; Kettenmann, H.

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

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Item Type:	Article
Title:	In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia
Creators Name:	Bakina, O., Conrad, T., Mitic, N., Vidal, R.O., Obrusnik, T., Sai, S., Nolte, C., Semtner, M. and Kettenmann, H.
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.
Keywords:	Flow Cytometry, Inbred C57BL Mice, Microglia, Patch-Clamp Techniques, Single-Cell Analysis, Physiological Stress, Animals, Hippocampus, Mice
Source:	PLoS ONE
ISSN:	1932-6203
Publisher:	Public Library of Science
Volume:	19
Number:	7
Page Range:	e0302376
Date:	11 July 2024
Additional Information:	Copyright: © 2024 Bakina et al.
Official Publication:	https://doi.org/10.1371/journal.pone.0302376
PubMed:	View item in PubMed

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