Deep single-cell RNAseq of postnatal day 7 microglia from wild type and Trem2 knockout brains

We generated single-cell RNAseq profiles of 369 microglia (183 from wild type and 186 from Trem2 knock-out), sorted in the gate CD45lowCD11+ or CD45lowCD11+Gpnmb+Clec7a+ (PAM enrichment), to compare gene expression of wild type vs. Trem2 knock-out microglia on the postnatal day 7. Single cells were FACS index sorted from the whole brain followed by Smart-seq2 library preparation and Illumina Nextseq (sequence depth > 1 million per cell). A total of 334 cells passed quality control for data analysis. Microglia in the Trem2 knock-out contained a similar PAM population with characteristic gene expression, suggesting that the presence of early postnatal PAM do not depend on TREM2. Single microglia were FACS sorted from male animals (C57BL/6J background) into 96-well plates. Libraries were prepared with a semi-automated Smart-seq2 protocol. Three QC criteria were used (Y=passed, N=not passed), and only cells that passed all three criteria were used for downstream analysis.本研究构建了369个小胶质细胞（microglia）的单细胞RNA测序（single-cell RNAseq）图谱，其中183个来自野生型（wild type）样本，186个来自Trem2基因敲除（Trem2 knock-out）样本。所有细胞均通过CD45lowCD11+或CD45lowCD11+Gpnmb+Clec7a+（PAM富集群）门控策略进行分选，旨在比较出生后第7天野生型与Trem2基因敲除小胶质细胞的基因表达差异。研究人员从全脑组织中通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）完成单细胞索引分选，随后采用Smart-seq2文库构建方案，使用伊诺华（Illumina）Nextseq测序平台进行测序，单细胞测序深度超过100万条序列。最终共有334个细胞通过质量控制，可用于后续数据分析。Trem2基因敲除组的小胶质细胞中存在表型特征相似的PAM群，其基因表达特征与野生型组一致，这表明出生早期的PAM群的存在并不依赖于TREM2蛋白。本研究从C57BL/6J背景的雄性小鼠脑组织中分选单个小胶质细胞至96孔板中，文库构建采用半自动化的Smart-seq2建库流程。本次分析设置三项质量控制标准（Y代表通过，N代表未通过），仅全部满足三项标准的细胞才可用于下游数据分析。