Cellular Neurosciences, Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
Humboldt Universität, Berlin, Germany.
PLoS One. 2024 Jul 11;19(7):e0302376. doi: 10.1371/journal.pone.0302376. eCollection 2024.
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.
我们应用贴壁序列技术从成年小鼠急性脑切片的皮质、海马体和胼胝体中单个小胶质细胞收获转录本。在用膜片钳技术记录膜电流后，通过吸管收集细胞质，并进行改良的 SMART-seq2 制备，随后进行测序。平均而言，在来自海马体、胼胝体和皮质的 113 个细胞中检测到 4138 个基因，包括小胶质细胞标志物如 Tmem119、P2ry12 和 Siglec-H。将我们的数据集与之前发表的来自 FACS 分离的小胶质细胞的单细胞 mRNA 测序数据进行比较表明，我们的贴壁序列数据集缺少两个细胞簇。FACS 特异性簇中标记基因的通路分析显示与小胶质细胞激活和应激反应相关。这表明在正常情况下，原位小胶质细胞缺乏与应激反应相关的转录本，并且通过机械解离和 FACS 分离小胶质细胞的过程会触发与激活和应激相关的基因表达。
