GSM8777006: scRNA-seq,AV_plus_P,CMO; Mus musculus; OTHER
At 5 days post infection, single cell suspensions were obtained from joint-associated tissue specimens by digestion in RPMI with 10% FBS, 15 mM HEPES, type I collagenase (Thermo; 2.5 mg/mL) and DNase (Sigma-Aldrich; 10 µg/mL) for 1 h at 37oC, followed by filtration through a 70 µm nylon filter. Cells were blocked with TruStain FcX™ PLUS (anti-mouse CD16/32, Biolegend) and then stained with TotalSeq anti-mouse hashtag antibodies (Biolegend B0302, B0304, B0306, B0308; one distinct hastag per mouse sample in each group following manufacturer's instructions) and fluorescently conjugated antibodies against cell surface antigens. After washing, individual samples were pooled according to experimental group and resuspended in sorting buffer (PBS with 1% FBS, 1mM EDTA, and 25 mM HEPES). Fluorescence activated cell sorting was performed using a BD FACSAria II flow cytometer (BD Biosciences). Sorted cell populations were pooled according to experimental group and resuspended in PBS with 0.04% BSA prior to processing for single cell RNA sequencing. RNA was extracted according to the 10x v3 3' Expression library kit. Libraries were prepared according to the 10x v3 3' Expression library kit, following manufacturer's instructions.