6 small intestinal macrophage subsets (CD4–Tim4–CD163–, CD4+Tim4–CD163–, CD4+Tim4+CD163–, CD4–Tim4–CD163+, CD4+Tim4–CD163+, CD4+Tim4+CD163+ ) from adult male C57BL/6J mice

To better determine functional heterogenity of small intestinal macrophages, 6 subsets of were sorted for RNA sequencing based on expression of CD4, Tim4, and CD163 25,000 - 50,000 small intestinal macrophages were isolated by fluorescence-activated cell sorting (FACS) from a single cell suspension, generated by mechanical disruption and enzymatic digest. Live CD45+Lin–(TCRb,B220,SiglecF,Ly6C,Ly6G)CD11b+MHCII+CD64+ cells were sorted into CD4–Tim4–CD163–, CD4+Tim4–CD163–, CD4+Tim4+CD163–, CD4–Tim4–CD163+, CD4+Tim4–CD163+, CD4+Tim4+CD163+ populations, directly into RLT buffer, frozen on dry ice and stored at –80C until RNA extraction using a Qiagen RNeasy micro kit