RNA-seq of small intestinal macrophages, neutrophils, CD103-CD11b+cDC and cryptopatch and ILF associated mononuclear phagocytes (CIA-MPh)

Cryptopatches (CP) and isolated lymphoid follicles (ILF) are evolutionary ancient lymphoid structures found in the intestines of all vertebrates. These structures are demarcated by a poorly characterised subset of mononuclear phagocytes (MPh), called CP/ILF-associated (CIA)-MPh. CIA-MPh expressed very high levels of lysozyme M (LysM), but they did not express Ly6G or CD64, normally associated with LysM-expressing neutrophils or macrophages, respectively. In order to understand the ontogeny and function of these newly identified MPh subset, we performed genome-wide transcriptional profiling of CIA-MPh and other cell types expressing LysM (i.e., neutrophils and macrophages) in the small intestine as well as of CD11b+ CD103- cDC2. Using Lyz2-GFP reporter mice, we developed a sorting strategy allowing us to highly purify neutrophils, macrophages, CD11b+ CD103- cDC2 and CIA-MPh. The RNA of each sample was extracted using the ImmGen protocol with phase lock tubes. The libraries were prepared using the SMARTer Ultra Low Input RNA kit (Clontech) according to the manufacturer's instructions. The material was enriched for poly-A sequences and single-end Next Generation Sequencing was performed on a HiSeq 2500 machine (Illumina).隐窝斑（Cryptopatches, CP）与孤立淋巴滤泡（isolated lymphoid follicles, ILF）是所有脊椎动物肠道中均存在的进化古老的淋巴结构。这类结构以一类尚未被充分表征的单核吞噬细胞（mononuclear phagocytes, MPh）亚群——即隐窝斑/孤立淋巴滤泡相关单核吞噬细胞（CP/ILF-associated, CIA-MPh）——作为边界标记。CIA-MPh高表达溶菌酶M（lysozyme M, LysM），但不表达通常分别与表达LysM的中性粒细胞、巨噬细胞相关的Ly6G与CD64分子。 为解析这一新发现的MPh亚群的个体发育与功能，我们对小肠内的CIA-MPh及其他表达LysM的细胞类型（中性粒细胞与巨噬细胞），以及CD11b+ CD103- 经典树突状细胞2型（cDC2）进行了全基因组转录谱分析。我们借助Lyz2-GFP报告基因小鼠，建立了可高效纯化中性粒细胞、巨噬细胞、CD11b+ CD103- cDC2与CIA-MPh的分选策略。 所有样本的RNA均采用相锁管（phase lock tubes）遵循ImmGen实验方案提取。文库构建依照制造商说明书，使用Clontech公司的SMARTer超低起始量RNA建库试剂盒完成。测序前对样本进行聚腺苷酸序列富集，并在Illumina公司的HiSeq 2500测序平台上开展单端下一代测序。