AM in vivo and ex vivo RNAseq

Here we show that alveolar macrophages maintain their core AM transcriptomic identity through culture expansion but show adaptations during culture expansion that are transient, reversible and fully restored upon transplantation into the alveolar niche. For expanded AMs (exAM), total RNA was extracted from exAMs after 2 months of culture. exAM were transplanted into 2 months old CD45.1 mice and analyzed 4 months post transplantation. In vivo AM subsets were sorted (Live, Singlets, SiglecF+, CD11c+, CD45.2+ or CD45.1+) from total lung homogenates directly into RNA lysis buffer (RLT buffer, Qiagen) for RNA extraction. RNA-seq samples were generated from a pool of 3 mice or exAM cultures in duplicates.