Illumina MiSeq paired end sequencing; Porcine nasal and lung macrophage subsets isolated by FACS and LCM show different transcriptomic profiles depending on tissue origin and location
To further investigate these macrophage subsets, total RNA sequencing was performed in parallel on two subsets from the nasal mucosa and lung macrophages. Macrophages were isolated by either fluorescent activated cell sorting (FACS; cf. bulk RNAseq) or laser capture microdissection (LCM; cf. LCM RNAseq) in combination with immunofluorescence staining against two macrophage markers, CD163 and Sialoadhesin (Sn). RNA was extracted using RNeasy Micro Kit (Qiageon; 74004) for bulk RNAseq subset, or using Single Shot Lysis Buffer for LCM RNAseq subset. Libraries were constructed using TruSeq Stranded mRNA Kit for bulk RNAseq subset, or using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara, 634413) for LCM RNAseq subset.