Illumina HiSeq 4000 paired end sequencing; Bulk RNA sequencing of CSF1-cultured bone marrow-derived macrophages from WT and Clec4a2-/- mice.
Bulk RNA sequencing of CSF1-cultured bone marrow-derived macrophages from WT and Clec4a2-/- mice.
CSF1-cultured macrophages were collected and sorted into CD11b+CD206high populations which then were separated by each genotype based on the expression of CD45.1 or CD45.2.  Bone marrow cells from CD45.1 and Clec4a2-/- (CD45.2) mice were co-cultured with 100 ng/ml CSF1 for 10 days.  Total RNA was isolated by using the RNeasy micro kit (Qiagen). Total RNA quantity and integrity were assessed using Quant-IT RiboGreen RNA Assay Kit (ThermoFisher Scientific) and high-sensitivity DNA tapestation (Agilent).   Purification of mRNA, generation of double stranded cDNA and library construction were performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) for Illumina with adapters and barcode tags (dual indexing). The final size distribution of the pool was determined using high-sensitivity DNA tapestation (Agilent), and quantification was determined by Quant-IT RiboGreen RNA Assay Kit (ThermoFisher Scientific).