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).