Gene expression analysis of predestined heterogeneity in neutrophil progenitor clones (ER-HoxB8 conditionally immortalized)

Transcriptional profiling of clonal neutrophil progenitor populations. Conditionally immortalized CD45.1 naive paired granulocyte-monocyte progenitors (GMPs) and their mature neutrophil counterpart were profiled in an in vitro cell system. Clones demonstrated an overall similar transcriptional profile within the GMP or neutrophil states, however, individual clones had several differentially expressed genes (DEGs) between each other at both the progenitor and mature cell stages. We utilized a technique of retroviral ER-Hoxb8 expression (Wang et al. Nature Methods 2006) to examine clonal granulocyte-monocyte progenitors (GMPs) and the corresponding mature neutrophil from a C57Bl/6-CD45.1-STEM mouse. The GMPs are will grow indefinitely in the presence of stem cell factor and beta-estradiol. Eight paired naïve granulocyte monocyte progenitors (GMPs) and neutrophil clones were processed for RNA-seq using the Qiagen RNeasy Plus Mini Kit (Qiagen) with DNaseI treatment.

克隆性中性粒细胞祖细胞群体的转录组分析。本研究在体外细胞体系中，对条件永生化的CD45.1阳性配对未致敏粒细胞-单核细胞祖细胞（granulocyte-monocyte progenitors, GMPs）及其成熟中性粒细胞对应物进行了转录组谱分析。各克隆在GMP或中性粒细胞状态下整体转录组谱较为相似，但在祖细胞阶段与成熟细胞阶段，单个克隆之间均存在多个差异表达基因（differentially expressed genes, DEGs）。我们采用逆转录病毒ER-Hoxb8表达技术（Wang等，《自然·方法学》，2006年），对来自C57Bl/6-CD45.1-STEM小鼠的克隆性粒细胞-单核细胞祖细胞（GMPs）及其对应成熟中性粒细胞开展了检测。在干细胞因子与β-雌二醇存在的条件下，GMPs可实现无限增殖。我们对8对未致敏粒细胞-单核细胞祖细胞（GMPs）与中性粒细胞克隆进行了RNA测序（RNA-seq），样本制备过程中使用Qiagen RNeasy Plus Mini Kit（Qiagen公司）并辅以DNaseI处理。