Bulk RNA-seq of adult homeostatic microglia from 4 brain regions

To compare microglial regional heterogeneity, we generated bulk RNA-seq profiles of postnatal day 60 microglia, sorted by TMEM119+ (also CD45lowCD11b+), from cortex (CTX), cerebellum (CB), hippocampus (HIP), striatum (STR) regions. For each sample, 3000 microglia were FACS sorted into RLT lysis buffer for total RNA extraction, followed by Smart-seq library preparation and Illumina Nextseq (sequence depth 10-20 million per sample). Consistent with our scRNA-seq data, samples from 4 regions were highly correlated (R>0.99), and individual samples did not cluster according to tissue origins, suggesting striking similarities between homeostatic microglia from different brain regions. Moreover, we could not detect any differentially expressed genes (FDR < 0.05) between regions from the bulk samples. These data suggest that classical adult microglia with homeostatic signatures (e.g. Tmem119), as the most dominant microglial population in the healthy brain, have little transcriptomic heterogeneity across brain regions. TMEM119+ microglia (3000 cells each sample) from a given region were FACS sorted from pooled male animal samples (C57BL/6N) into RLT lysis buffer in Eppendorf tubes. Three replicates were done for each region. Libraries were prepared following the Smart-seq protocol (v4 ultra low input RNA kit). All samples were barcoded and pooled together for Illumina Nextseq sequencing.为比较小胶质细胞的区域异质性，我们构建了出生后第60天小胶质细胞的批量RNA测序（bulk RNA-seq）图谱：这些细胞通过TMEM119阳性（同时为CD45lowCD11b+）分选自皮层（CTX）、小脑（CB）、海马体（HIP）以及纹状体（STR）区域。每份样本取3000个小胶质细胞，经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）后置于RLT裂解液中进行总RNA提取，随后开展Smart-seq文库制备，并使用Illumina Nextseq进行测序（每样本测序深度为1000万至2000万条序列）。 与我们的单细胞RNA测序（scRNA-seq）数据一致，来自4个脑区的样本相关性极高（R>0.99），且单个样本并未按组织来源聚类，这提示稳态状态下不同脑区的小胶质细胞间存在显著的转录组相似性。此外，我们未在批量样本的脑区间检测到任何差异表达基因（错误发现率（False Discovery Rate, FDR） < 0.05）。 上述数据表明，以稳态特征标志物（如Tmem119）为代表的经典成年小胶质细胞，作为健康大脑中占绝对优势的小胶质细胞群，其跨脑区的转录组异质性极低。 来自特定脑区的TMEM119阳性小胶质细胞（每份样本3000个细胞），从混合的雄性C57BL/6N小鼠样本中经FACS分选后，被置于Eppendorf管的RLT裂解液中。每个脑区设置3个生物学重复。文库构建遵循Smart-seq建库方案（v4 ultra low input RNA kit，即Smart-seq v4超低起始量RNA建库试剂盒）。所有样本均添加了条形码并混合后，使用Illumina Nextseq完成测序。