GSM7156474
Sorting of synovial macrophages was carried out from the knee cell suspensions stained as paper described. Lining macrophages were defined as CD45+, Linage- (NK1.1, CD3, CD19), CD11b+, F4/80+ and VSIG4+ cells, whereas sublining macrophages were CD45+, Lin-, CD11b+, F4/80+ and VSIG4-. Two hundred macrophages were sorted through 70-µm nozzle directly into 2.2 µl of lysis buffer (composition detailed below) using BD FACS Aria III. Two hundred lining and sublining macrophages from 3 WT and 3 IRF5KO mice, isolated from PBS and mBSA injected knees, were sorted into PCR tubes containing 2.2 µl lysis buffer (0.8% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor (Clontech)). Samples were transferred into 96-well skirted PCR plate, sealed with an adhesive PCR plate seal (Thermo Fisher), and transferred to Oxford Genomics Centre on dry ice to be sequenced. Briefly, the preparation of libraries was done using NEBNext® Ultra-low/single cell (Smarter-like) protocol (New England BioLabs).